Note: Descriptions are shown in the official language in which they were submitted.
CA 02229~1~ 1998-03-13
EXPRESSION OFA FOAMY YIl~US ENVEL(,'~P~ PROTEIN
The foamy viruses (FV) subgroup of retroid viruses has attracted scientific
5 interest because of their unique replication strategy and because of their potential
use as gene transfer vectors (3 5). It has been proposed that FVs may be ideal tools
for the development of a gene delivery system due to specific properties of this virus
group, such as the absence of FV antibodies in the human population, the benign
course of natural FV infections, their very broad host cell range, and an extended
packaging limit due to the size of the FV genome (4, 30, 32). However, limited
knowledge of the molecular biology of this virus group has so far not allowed the
development of safe pack~ging cell lines and vectors, such as those that have been
derived for murine retroviruses, among others (27). For instance, the FV is a DNA
virus with a complex genome organization. In addition to LTRs (Long Terminal
15 Repeat), a pack~ging region and gag, pol, env genes, it also comprises several genes
such as bell, bel2, bel3, bet, beo and bes located between env and 3'LTR. The env
gene encodes a 1~0 kDa glycosylated precursor that is cleaved giving rise to thesurface (SU) and transmembrane (TM) subunits (see Fig. 1 and 4). The TM subunit
includes in its 3' part a transmembrane anchor dornain (A in Fig 4) composed of
20 hydrophobic residues which is followed by a cytoplasmic tail. Furthermore, FVs
express their Pol protein from a spliced mRNA independently of the Gag protein,
and the mech~nism of FV genome pack~ging and particle assemb!y, as well as the
significance of high amounts of reverse transcribed DNA. in the extra-cellular
particle are largely unknown (10, 18, 39). Other unique features include the nuclear
25 localization of the Gag precursor protein (31, 40) and the predominant budding illtO
intracytoplasmic vesicles which may be a consequence of the retention of the Envprecursor protein in the ER (13).
Moloney retrovirus-based gene transfer vectors are currently the main
vehicles for high efficiency stable gene transfer into a wide variety of cell types (20).
CA 02229~1~ 1998-03-13
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Major limitations of this vector system are the restricted host cell range and the
inefficient infectivity for some human cells (reviewed in (1)). Recently, several
methods USillg the pseudotyping with foreign envelope proteins, such as the
ves,icular stomatitis virus (VSV) G glycoprotein (6, 38) or the gibbon ape leukemia
5 vims (GALV) envelope protein (2, 34) have been shown to overcome these
disadvantages.
However, the expression of VSV-G for example is highly toxic for the
producer celis and has prevented the generation of stable VSV-G packaging cells
line (8, 22, 37).
The invention concems constructs for the expression of a protein comprising
at least a modified FV envelope protein.
The preferred FV according to the present invention is the human foamy
vims (HFV), but others may be used (e. g. Simian FV).
The modification may consist in at least a mutation, deletion, substitution
15 and/or addition of one or several amino acid (aa) of said modified FV envelope
(env) protein or a combination thereof. Such modification(s) is preferably located
into the cytoplasmic tail. Advantageously, a modified FV envelope protein is
truncated at aa 975 or, more preferably, 981. The truncation may extend up to the
stol) codon or alternatively comprise before the stop codon one or several residues
20 opti.onally from the original FV env protein.
Furthermore, a construct of the invention may express a mature modified FV
envelope protein or a precursor thereof or a chimeric protein resulting from ~hefusi~n of sequences of various origins. In a particularly preferred embodiment, the
modified FV env protein in use in the present invention is a fusion protein which
25 furt:herrnore comprises all or preferably a part of a non-FV envelope protein.
Examples of suitable non-FV viruses include avian retroviruses, bovine retroviruses,
feline retroviruses, murine retroviruses such as Murine Leukemia Virus (MuLV) and
particularly Moloney MuLV (MoMuLV), Friend Murine Leukemia Virus (FrMuLV)
especially strain FB 29, Murine Sarcome Virus (MSV), primate retroviruses such
CA 02229~1~ 1998-03-13
as GaLV, VSV or lentiviruses such as HIV (Human Immunodeficiency Virus) or
SI'~r (Simian Immunodeficiency Virus)
The fusion betwen FV and non-FV env proteins can be made at di~rerent
locations. Fusions within the TM subunit are advantageous. According to a first
5 alternative, the fusion is within the transmembrane anchor domain of said FV and
non-FV envelope proteins. A prefered example is a protein that comprises the
extracellular domain and the 5' part of the transmembrane anchor domain of the
HFV envelope protein and the 3' part ofthe tr~ncmembrane anchor domain and the
cytoplasmic domain of the non-FV envelope protein, particularly of the SIV
10 en~elope protein.
A second alternative is that the fusion is within the cleavage site of said FV
and non-FV envelope proteins. A prefered example is a protein that comprises theSU domain and all or part of the cleavage site of the HFV envelope protein and all
or part of the cleavage site and the TM domain, comprising the tr~nsmembrane
15 anc~hor domain and the cytoplasmic domain, of the non-FV envelope protein,
particularly ofthe SIV envelope protein. The replacement ofthe cleavage site ofthe
FV envelope protein by its equivalent from the non-FV envelope protein is also
envisageable.
Another alternative is that the fusion is at the junction between the
20 transmembrane anchor domain and the cytoplasmic domain or within the
cytoplasmic domains of said FV and non-FV envelope proteins. A prefered example
is a protein that comprises the extracellular domain, the transmembrane anchor
domain and all or part of the cytoplasmic domain of the HFV envelope protein andall or part of the cytoplasmic domain of the non-FV envelope protein, particularly
25 of the SIV envelope protein.
In a particularly preferred embodiment, a protein according to the invention
consists in HFV protein envelope which all or part of the cytoplasmic domain is
replaced by all or part of a cytoplasmic domain of a non-FV viral envelope protein,
especially of a MuLV envelope protein. Advantageously, the fusion protein consists
CA 02229~1~ 1998-03-13
in the fusion of a MuLV cytoplasmic domain to a modified HFV envelope protein.
The MuLV cytoplasmic domain in use in the present invention may be processed or
unprocessed. ~Processed~ means that it contains the cleavage site normally
rec ognized by the corresponding retroviral protease and ~unprocessed~ that it does
not contain it or that it is not functional (mutation, deletion or truncation).
The preferred construct of the invention is the one allowing expression of
the fusion protein design~ted hereinafter HFV ~2 MuLV.
Alternatively, a prefered protein according to the invention consists in a
HFV envelope protein in which all or part of the cytoplasmic tail is replaced by all
10 or part of a cytoplasmic domain of a SIV envelope protein. There exists two
versions ofthe SIV envelope protein: a long form having a cytoplasmic tail of 164
amino acids which is present in SIV particles replicating in the natural host, the
rhesus monkey (Macaca mulatta) and a short form cont~inin~ only 18 amino acids.
This short form is selected for when virus isolated from the monkey are cultured on
15 hurnan cell lines (42).
It is also possible that the construct of the invention is mutated in the donor
and~or acceptor splicing sites naturally present in the FV env protein encoding
sequence.
The construct of the invention may include regulatory elements to allow
20 transcription and translation of the sequence coding for the modified FV env
protein. In particular, a suitable promoter may be linked up~tl~alll from the FV env
encoding sequence in an operative way by conventional recombinant techniques.
Such a promoter may be of prokaryote, eukaryote or viral origin and may be
constitutive or re~ ted. Such regulatory elements are well known in the art.
It is also in the scope ofthe invention that the construct ofthe invention may
additionally comprise a selection gene enabling detection and isolation of the cells
exp:ressing the modified FV env protein. In the context of the invention, the
selection gene may be under the transcr,ptional control of the promoter driving
e,.p,.ession of the modified FV env protein resulting in a bicistronic transcript or
CA 02229~1~ 1998-03-13
under the control of an additional promoter region. The possible selection genes are
numerous, for example neo gene conferring resistance to antibiotic G4 18,
dihvdrofolate reductase (dhFr) gene, puromycin acetyl transferase (pac) gene or
xanthine phosphoribosyl transferase (gpt).
The construct of the invention may be inserted in any appropriate vector, a
viral vector (e.g. a retroviral vector) or a plasmid. The choice of the appropriate
vector is large and within the capabilities of the man skilled in the art. Such a vector
may be integrative or not. To decrease the possibility to generate replication-
competent viral particles, it is advantageous that the construct lack any retroviral
10 LTR and paçk~ging region.
The invention also concerns fusion proteins as expressed by the above
expression constructs as well as pseudotyped viral particles comprising a FV envprotein. This latter may be derived from a native FV env protein, a part thereof or
a modified one. In a preferred embodiment, the pseudotyped viral particle at its15 surfàce comprises a modified FV env protein as expressed by a construct according
to the invention. The pseudotyped viral particle of the invention may be generated
upon transfection of a recombinant viral vector into a complementation cell line. The
tec~mology is conventional and desclibed in numerous prior art documents. A viral
vecltor in use in the present invention comprises preferably at least a 5' LTR, a
20 paçk~ging region and a 3' LTR derived from any retrovirus such those cited
previously and a gene capable of expressing a ribozyme, an anti-sense RNA
molecule or a mRNA to further produce a polypeptide of interest. Of particular
interest, are therapeutic polypeptides, including but not limited to cytokines (IL-2,
IFN a, ~ or y), Herpes Simplex Virus type 1 (HSV- 1) thymidine kinase (TK), Cystic
25 Fibrosis Tr~n.~" ~e"ll-l~1e Conductance ~eg~ tQr (CFTR), Dystrophin, coagulation
Factors ~FVIII, FIX, ...), tumor-associated antigens (MUC-1, HPV antigens),
antibodies, immunotoxines, anti-HIV drugs, growth factors (Fibroblast Growth
Factor FGF, Vascular Endothelial Growth Factor VEGF), apoptosis inducers
(Ba~...), apoptosis inhibitors (Bc12, Bclx...), cytostatic agents (p21, pl6, Rb), epo
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- 6 -
lipoproteins, nitrix oxid synthetase (Nos), oxigen radical scaveyers (SOD,
catalase...), tumor suppressor products (p53, p73) and markers. This list is notlimiltative. Furthermore, the virai vector in use in the present invention may transfer
one or more genes in a native, tmnc~ted, mutated or hybrid form. The gene(s) is
5 placed under the control of elements allowing its expression in an eukaryotic cell.
Such elements includes a promoter, which may be from any origin (retroviral LTR
or internai promoter). It may be constitutive or responsive to cell or tissue-specific
factors.
Another object of the invention is relating to complementation cell line
10 perrnitting the production of the pseudotyped virai particles and the method of their
preparation.
The invention further concerns complementation cell line comprising a
construct of the invention.
Preferably, said complementation cell line comprises a construct of the
15 invention characterized in that fusion is at junction between the transmembrane
anchor domain and the cytoplasmic domain or within the cytoplamic domains of said
FV and non-FV envelope proteins and in that the expressed protein comprises the
extracellular domain, the transmembrane anchor domain and all or part of the
cytoplasmic domain of the HFV envelope protein and ail or part of the cytoplasmic
20 domain of the non-FV envelope protein, particulariy of the SIV envelope protein.
In another p~felled embodiment, the said complementation cell line
comprises a construct of the invention for the expression of a protein characterized
in that the protein is ~V~2 MuLV.
The complçrnent~tion cell line ofthe invention may derive from any cell and,
25 particularly, eukaryotic cell. One may envisage murine cell lines, pharmaceutically
acceptable cell lines (Vero, CHO, ...) or human cell line such as 293 or A549. It
may be generated by transfection of a construct according to the invention alongwith a first selection gene. The highest env producer cells are then screened for
e~cyl ession of high levels of FV env protein by immunodetection using antibodies
CA 02229~1~ 1998-03-13
against FV env, Western blot, FACS (Fluorescente Activated Cell Sorter) or any
other method. Alternatively the complementation cell line of the invention, may also
comprise a construct expressing a viral gag / pol gene, more preferably of MuLV,FB 29, SIV or ~V along with a second selection gene di~elelll from the first one.
5 Preferably, the env and gag / pol genes are carried by separate expression vector
lacking LTR and p~ck~ging region. The selection and screening steps are repeatedto select a env producing clone which further expresses gag / pol expression
product.
A complementation cell line of the invention may be used to package
10 recombinant viral vector. The titer may be tested using a conventional viral vector
expressing a third selection gene different from the previous ones or a marker gene
(e.~;. Lac Z). As a result, cells producing high titers of pseudotyped viral particles
are selected and can be cultured to supply a stable complementation cell line. The
cells may also be tested transiently as usually performed and described hereinafter.
According to another aspect of the invention, it is also provided a method
for preparing a pseudotyped viral palticle of the invention. Such a method comprises
the act of (1) introducing a recombinant retroviral vector into a complementation
cell line of the invention, (2) culturing said complementation cell line under suitable
conditions permitting production of the said pseudotyped viral palticle and (3)
20 recovering the resulting pseudotyped viral particle from cell culture.
Preferably, the pseudotyped viral particle is recovered from cell culture
supernatant but a cell Iysis step may also be considered. The pseudotyped viral
parl.icle may also be further purified by conventional technGlogy (e.g.
ultracentrifugation on sucrose or ClCs gradient). ~dvantageously, the pseudotyped
25 viral particle thus produced is able to infect (preferably in the absence of polycation
such as polybrene) a wide variety of cells and optionally to resist to inactivation by
human serum.
According to another aspect of the invention, it is also provided a
m~rnm~ n host cell infected by the pseudotyped viral particle of the invention or
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-- 8 --
obtainable by a method of the invention. Such a host cell includes ~vithout limitation
human epithelial, pulmonary, mllsc~ r, hepatic, haematopoietic cells, fibroblastes
and Iyrnphocytes.
A pseudotyped infectious particle as well as a m~mm~ n cell of the
5 invention may be applied in the prevention or treatment of various diseases, as a
vaccine or a therapeutic agent.
It is also the scope of the invention to provide for a pharm~ceutical
composition comprising a therapeutic,ally or prophylactically effective amount of a
pseudotyped viral particle of the invention or obtainable by a method of the
10 invention as well as a m~mm~ n cell of the invention as a therapeutic agent. Such
a pharmaceutical composition may be produced in a conventional manner. In
par~:icular, the particle or the m~mm~ n cell of the invention may be combined with
appropriate substances well known in the art, such as a carrier, diluent, adjuvant or
excipient. The particular formulation of the pharmaceutical composition depends on
15 various parameters, for example the polypeptide of interest to be expressed, the
desired site of action, the method of administration and the subject to be treated.
Such a formulation can be determined by those skilled in the art and by conventional
knowledge.
Typically, the pseudotyped particles are prepared as a solution in an
20 acceptable diluent~ such as sa]ine, phosphate-buffered saline or other
pharmaceutically acceptable diluent. The route of inoculation may be intraveinous,
intramuscular, subcutaneous, intradermal, intrapulmonar, intratracheal, intragastric
and intratumoral. The dose may be unique or repeated. The quantity will vary
depending upon inciividuals (weight, sex, age, medical conditions....) and the disease
25 to t:reat. In general, it is desirable to provide the pseudotyped viral particles of the
invention in the range of from about 104 to about 10l3 plaque forming units
(pfu)/dose, avantageously from about 105 to about 101~ and preferably from about
106 to about 109. The composition of the invention can be introduced into a
m~mm~l (human) ex vivo by prealable exposure oftarget cells to the pseudotyped
CA 02229~1~ 1998-03-13
viral particles before introduction ofthe transduced cells into a m~mm ll, or injected
in vrvo into the affected tissue, in the circulation or locally.
In a last embodiment of the invention, it is also provided a method of
treating a genetic disorder or a disease induced by any pathogenic gene, such as5 cancer or a virally-induced disease, which comprises ~tlmini.~tçring a therapeutically
effective amount of a pseudotyped viral particle or a m~mm~ n cell of the invention
to a subject in need of a treatment.
These and other advantages of the subject invention will be apparent from
the following examples and attached drawings. These embodiments do not represent10 the fi~ll scope of the invention.
In particular, incorporation of human foamy virus (HFV) envelope proteins
into murine leukemia virus (MuLV) particles was studied in a transient transfection
paçk~gin~ cell system. We report here that wildtype H~V envelope protein can
pseudotype MuLV particles, albeit at low efficiency. Complete or partial removal15 of the HFV cytoplasmic tail resulted in an abolishment or reduction of HFV
mediated infectivity, implicating a role of the HFV envelope cytoplasmic tail in the
pseudotyping of MuLV particles. Mutation of the ER retention signal present in the
HFV envelope cytoplasmic tail did not result in a higher relative infectivity ofpseudotyped retroviral vectors. However, a chimeric envelope protein, cont~ining20 an unprocessed MuLV envelope cytoplasmic domain fused to a truncated HFV
envelope protein, showed an enhanced HFV specific infectivity as a result of an
increased incorporation of chimeric envelope proteins into MuLV particles.
Brief description of the drawings
Fi~ure l
Schematic illustration of the ~IFV envelope expression constructs.
The extracellular, membrane spanning (also desi~n~ted tr~n~membrane
anchor domain A), and cytoplasmic domains of the TM components of the ~V
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- 10-
(open boxes) and the MuLV (shaded boxes) envelopes are shown according to (11,
24). The amino acid sequence of the wildtype HFV and MuLV proteins are given
below the schematic illustration. The amino acid positions in the HFV envelope
constructs are marked on the ruler. The location of the sequence motif in the
5 cytoplasmic domain of the HFV envelope, responsible for ER retention (13) is
indicated as a black box, the mutated sequence as a striped box. The cleavage site
of the MuLV protease in the full length cytoplasmic domain of the MuLV envelope
protein is indicated by two inverted arrows
10 Fi~ure 2
Infcctivity of MuLV particles pseudotyped with different envelope proteins.
N~I3T3 (shaded bar) or QT 6 (solid bar) cells were infected with different
pseudotyped MuLV particles generated by transient transfection of 293T cells.
Forty-eight hours after transduction the percentage of GFP expressing cells was
15 qu~ntit~ted by FACS analysis. The mean fiuorescence of GFP expressing cells was
lO0 to 300 fold above those of mock infected cells. The individual envelope
constructs used for pseudotyping are indicated on the y-axis of the graph. The mean
percentage of GFP expressing cells for each construct is shown on the x-axis with
the corresponding standard deviation. ~ndividual constructs were tested 2-6 times.
Fi~ure 3
Neutralization of ~IFV envelope specific infectivity.
MuLV particles pseudotyped with different envelope proteins, as indicated
on the y-axis, were generated by transient transfection of 293T cells. Supernatants
25 (l ml) were incubated with anti-HFV specific chimpanzee serum (1:60) (solid bar)
or human serum (1:60) from a healthy individual (shaded bar) for l hour at 37~C,prior to the addition to NIH3T3 (A) or QT-6 (B) cells. The supernatant was
aspirated four hours later, and replaced with fresh growth medium. Forty eight
hours after transduction, the percentage of GFP expressing cells was determined as
CA 02229~1~ 1998-03-13
described in the legend of Fig. 2. Ihe experiment wa.s carried out twice with a
neutralizing monkey serum and in addition, with an anti-HFV surface rabbit senJm(data not shown) resulting in a similar relative inhibition of the infectivity of H~V
envelope pseudotyped retroviral vectors.
Fi~ure 4
Schematic representation of the chimeric HFV/SIV envelope constructs.
HFV WT and SIV are wild type versions of the human foamy virus and
simian immunodeficiency virus (molecular clone mm25 1 ) envelope genes. Domains
10 from HFV are in white and domains from SIV in grey. Cleavage site separing SUand TM domain is indicated with a vertical line. RRE represents rev responsive
element and A the transmembrane anchor domain. The extracellular domain in 5' ofthe transmembrane anchor domain and the cytoplasmic domain in 3' of the
transmembrane anchor domain are also indicated. Env 1 to 9 are represen~alion of15 the chimeric ~V/SIV env constructs comprising fusion with long version (Env 1,
3 and 5) or short version (Env 2, 4, 6, 7, 8 and 9) of SIV envelope.
Fi~ure 5
Sequence of the ~IFV, S~V and chimeric env genes fl~nkin~ the fusion sites and
20 amino acid sequences of the long and short cytoplasmic tails from SIV env.
A. illustrates the transmembrane anchor domains of HFV and SI~ mm25 1
env genes and the fusion in the transmembrane anchor domain of ~IFV and SIV
mm25 1 (Chim): identical arnino acids in the HFV and SIV envelopes are in bold,
the transmembrane anchor domain sequences of HFV and SIV envelopes are
25 underlined. B. illustrates the fusion at the cleavage site between SU and TM
domains of HFV and SIV mm25 1 envelopes: consensus sequence of the cleavage
site is in bold, the cleavage site is indicated by a space in the amino acid sequence.
C. illustrates the substitution of the cytoplasmic tail of ~V envelope with the
cytoplasmic tail of SIV mm25 1 envelope: the transmembrane anchor sequences are
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- 12 -
underlined, (141) indicates the last 141 amino acids from the SIV long cytoplasmic
tail. D. amino acid sequences of the long and short cytoplasmic tails from SIV
envelope: transmembrane anchor domains are underlined, * indicates a stop codon,(14:1) indicates the last 141 amino acids from the SIV long cytoplasmic tail.
Fi~ure 6
Destruction of the SIV mm 251 env internal 3' splice site.
The amino acid sequence shown on top is from the SIVmm251 env gene, *
indicates the stop codon of the hllm~ni7ed env gene. A indicates the G to A
10 mutation reslllting in the destruction of 3' splice site. Tat/Rev exon 2 starts at the
sequence TAGACT but the reading frame is different from the env reading frame.
The present invention will IIOW be illustrated in the following and non
limit~,ting examples.
EXAMPLES
All const~uctions are made by using standard recombinant DNA techniques
such as those described in T. Maniatis et al., Molecular cloning: a laboratory
20 rmanual, Cold Spring Harbor, NY 1982. The cell lines are accessible by the culture
collections such as ATCC and cultured by standard conditions (NIH3T3: CRL-
165g, Mv.1.Lu CCL64, HT 1080 CCL 121, BHK 21 CCL 10, QT 26 CRL 1708
and 293 CRL 1573). The sequence of the HFV env protein has already been
published and is available in EMBL data base (accession number 407725).
EXAMPLE 1: EIFV/MLV chimeras.
1. Generation of FV env expression construct
An eukaryotic expression construct for the envelope gene of the human FV
isolate (HFV) was generated by inserting a 3076 bp AflII/EcoRI ~ragment of the
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- 13 -
HFV proviral clone pHSRVI (28), cont~ining the full-length env open reading frame
(ORF), into the pCDNA3 (Invitrogen) vector. This construct was design~ted
pCE~FV wt and used to generate the mutant and chimeric HFV envelope proteins
depicted in Fig. l. Briefly, truncated or chimeric env constructs were made by using
the polymerase chain reaction on HFV and/or MuLV env genes as templates and
oligonucleotides incorporating the desired mutations. The mllt~nts were insertedinto the basic vector described above and sequenced to exclude off-site mutations.
Three mutant HFV envelope constructs were generated. pCHFV ~1 and pCHFV ~2
code for HFV envelope proteins truncated at aa 975 or 981, respectively. pCHFV
10 ~2 has a C-terminal Arginine added, not present in the original HFV env sequence.
According to the HFV envelope domain structure proposed by Flugel et al. (11) the
truncations resulted in a complete (pCHFV ~l) or partial removal (pCHFV ~2) of
the cytoplasmic domain. Finally, the pCHFV SSS construct produces an HFV
envelope protein that has the triple Iysine motif (aa 984-986) at the C-terminal end
15 of the cytoplasmic tail of the transmembrane (TM) protein replaced by serine
residues. This sequence motif has been shown to be responsible for the ER retention
ofthe HFV envelope (13, 14).
In total 6 chimeric envelope proteins were constructed by C-terminal fusion
of sequences coding for the unprocessed or processed cytoplasmic domain of the
20 MuLV envelope protein (16, 17). pCHFV ~IMuLVR-, pCHFV ~2MuLVR- and
pCHFV SSSMuLVR- encode fusion proteins consisting of the 3 mutations
described above and a processed Mul,V envelope cytoplasmic domain (aa 634-649),
whereas pCHFV ~lMuLV, pCHFV ~2MuLV and pCHFV SSSMuLV encode the
respective fusion proteins cont~ining an unprocessed MuLV envelope cytoplasmic
25 domain (aa 634-665) at the C-terminus.
The expression constructs for the MuLV gag/pol (pHIT60), the ecotropic
(pHlT123) and amphotropic (pHIT456) MuLV envelope were kindly provided by
A. ~ing~m~n (33). The retroviral vector SFG GFPS65T contains the hum~ni7ed
ORF of the green fluorescent protein (7) (a gi~ of M. Vogel) inserted into the
CA 02229=.l=. l998-03-l3
-14-
cloning sites of the MuLV based retroviral vector SFG (5, 22), whereas MFG. S
NLS-LacZ (22) contains the ,~-galactosidase gene fused to the SV40 nuclear
localization signal (NLS) (a gifl of R. Mulligan). The VSV-G expression construct
was generated by inserting a 1.6 kb EcoRI fragment from plasmid pSVGL-I (29) (a
5 gift of J. Rose~ cont~ining the VSV-G ORF, into the pHIT vector.
- 2. Infectivity of MuLV particles pseudotyped with various ~IFV env proteins
Recombinant retroviral particles were generated using the pHlT pac.lc~ging
system essentially as described previously (33). Briefly, 293T cells (9) were
10 transiently co-transfected with an expression construct for MuLV gag / pol
(pHlT60), the MuLV based retroviral vector SFG GFPS65T, and the di~elenl
envelope expression constructs described above. Viral supernatants were harvested
48-72 hours after transfection. Supematants from independent transfections with the
same plasmids were pooled, filtrated (0 4511m pore size), polybrene was added to15 a final concentration of 8 lag/ml, and the supernatants were used immediately or
stored at -80~C until use. Target cells expressing the GFP protein after retroviral
transduction were identified by FACS analysis on a FACScan, and the number of
positive cells were quantitated using the LysisII and CellQuest Software package(Becton Dickinson).
Initial experiments using the pC~V wt expression construct showed that
MuLV particles can be pseudotyped with the HFV wt envelope protein and are able
to transduce NIH3T3 cells, albeit at low efficiency (Fig. 2). The HFV envelope
prol:ein contains a signal sequence in its cytoplasmic domain that leads to a retention
in the ER of expressing cells (13, 14). Therefore, three conshucts, pCHFV ~1,
pCHFV ~2, and pCHFV SSS, coding for cytoplasmically truncated or mutated HFV
envelope proteins were examined to determine the influence of the cytoplasmic
domain of the HFV envelope and its ER retention on the pseudotyping efficiency.
The complete (pCHFV ~1) or partial removal (pCHFV ~2) of the cytoplasmic
domain of the ~V envelope results in an abolishrnent or reduction of the already
CA 02229~1~ 1998-03-13
- - 15 -
low pseudotyping activity observed for the wildtype protein (Fig. 2). Mutation of
the cytoplasmic ER retention signal (pCHFV SSS) has previously been shown to
increase cell surface expression of the HFV envelope protein (13). However,
pseudotyping of viral particles with such a mutant protein also did not result in
5 higher infectivity of these viruses (Fig. 2).
Since removal or modification of the H~V cytoplasmic domain failed to
increase the infection efficiency of' pseudotyped virus, a second approach has
subsequently been used to test whether the replacement of the HFV cytoplasmic
domain by the MuLV cytoplasmic domain, or the fusion of the MuLV cytoplasmic
10 domain to a modified full-length HFV envelope would have the desired effect. The
cytoplasmic domain of the MuLV envelope was shown to be processed by the
MuLV protease in the viral particle (16, 17). Expression of an already processedforrn of the MuLV envelope protein in cells resulted in the formation of large
ml]ltinucleated syncytia and a decrease of viral infectivity (24, 26). Therefore, C-
15 terminal fusion proteins ofthe three mllt~nts described above and the processed(MuLVR-) or the unprocessed (MuL,V) cytoplasmic domain of the MuLV envelope
protein were generated and particles pseudotyped with these chimeric envelope
proteins were tested for their infectivity on NIH3T3 cells. Interestingly, viruses
pseudotyped with one mutant, the HFV ~2MuLV protein, showed a 10-20 fold
20 higher infectivity than particles pseudotyped with the wildtype HFV envelope
protein (Fig. 2). This increase in infectivity through the H~V ~2MuLV protein was
not specific for NIH3T3 or murine cells, as similar results were obtained for the
quail fibroblast cell line QT-6, which is not infectable by viral particles coated with
MuLV envelope proteins (Fig. 2). In these cells the infectivity of particles
25 pseudotyped with the HFV ~2MuLV envelope protein was consistently higher thanthose pseudotyped with the VSV-G protein. All other proteins analyzed gave rise
to pseudotyped viruses with lower or similar relative infectivity when compared to
wildtype HFV envelope on both cell lines (Fig. 2). In addition, chimeric HFV
envelope proteins cont~ining a processed MuLV cytoplasmic domain showed a
CA 02229~1~ 1998-03-13
- 16-
higher fusion activity than the corresponding proteins having an unprocessed MuLV
cytoplasmic domain upon expression in L929 cells by retroviral vectors (data notshown). This result is in accordance with data showing that the cytoplasmic domain
of the MuLV envelope can control the fusion activity of foreign envelope proteins,
5 such as the simian immunodeficiency virus (SIV), when expressed as a chimeric
envelope protein (36). Furthermore, supernatants cont~ining a retroviral vector
coding for a nuclear localized ,~-galactosidase protein pseudotyped with the dirrele~"
envelope proteins were titrated on cell lines of-various species (Table). More
precisely, target cells (1 x 104 cells/well) were plated 24 hours prior to infection with
10 serial dilutions of supernatants of transfected 293T cells. Fourtyeight hours after
infection the numbers of blue foci were counted in duplicates and the titers
calculated. The values of the duplicates were within a 3-fold range. The resultsshown are a representative of two independent titrations on the cell lines indicated
using for all cell lines cell free supernatants from the same transfections, with
15 reproducible relative titers in both experiments. Supernatants containing
pseudotyped particles were titrated UI) to 6 times on NIH3T3 cells with reproducible
results. Retroviral particles pseudotyped with the IIFV wt envelope protein or the
HFV ~2MuLV chimera were able to infect celis of human, mink quail and hamster
origin, in addition to murine cells (Table 1). The titers of retroviral vectors
20 pseudotyped with the HFV ~2MuLV envelope protein were ~-3 5 fold higher than
those pseudotyped with the wildtype :HFV envelope protein depending on the target
cells used.
CA 02229515 1998-03-13
- 17-
~ , O o
C.~ ~ ~ -- X X X
~ O~ V V ~ ~ ~
-
_,
o ~ o o o o o
X X X X X
O ~ ~ ~ ~~
o P VC'~
.
_
o .-- o '~
~- 'J oo O O o o
~ o o-- _ ~
o~ ~ ~ V
o
_ o
L ~ O -- _ ,_, ~
C~--1 -- X X X X
~
$ ,~ O O O O O
X X X X X
O ~ ~ ~ t-- ~ O
C~
U~
O ~ O O O O O
X X X X X
~ _ ~O ~ O
CC Z ~ ~
--
o .
e .o c~
_l ~ ~ ;> ;>
CA 02229~l~ l998-03-l3
- 18 -
3. Neutralization of the infe~livily of EIFV env pseudotyped particles by HFV
specirlc antisera
To confirm that the infectivity of MuLV particles pseudotyped with di~t en~
5 HFV envelope proteins was specific for the HFV envelope, pseudotyped particleswere prein~lb~ted with an anti-HFV specific chimp~n7ee serum prior to the addition
to target cells (3). The infectivity of viral particles pseudotyped with the
amphotropic MuLV envelope or the VSV-G protein was not reduced by
preincubation with the HFV specific antiserum when compared to the preincubation10 of these viruses with normal heat inactivated human serum (Fig. 3) or mock
incubated viral particles (data not shown). In contrast, the infectivity of particles
pseudotyped with the wildtype HFV envelope protein or the HFV ~2MuLV chimera
was completely abolished by the preincubation with the HFV specific antiserum but
not the human control serum (Fig. 3). This neutralization of HFV envelope specific
infectivity was observed for NIH3T3 (Fig. 3A) and QT-6 (Fig. 3B) cells. A similar
specific neutralization of viral particles pseudotyped with HFV envelope proteins
was obtained in experiments using a rabbit serum raised against the baculovirus
expressed SU domain of the HFV envelope protein (data not shown).
20 4. Expression and particle incorporation of IIFV env proteins
The expression and incorporation of the dirrele~ll HFV envelope proteins
into MuLV particles was determined by radioirnmunoprecipitation analysis (l~IPA)oftransiently transfected 293T cells. Forty-eight hours after addition ofthe DNA(pHIT60, SFG GFPS65T and various env constructs), cells were metabolically
25 labeled with [35S]methionine for approximately 20 h. Viral particles present in the
supernatant were pelleted by centrifugation at 25 000 rpm through a 20 % sucrosecushion prior to solubilization in Iysis buffer. Subsequently, the samples were
submitted to immllnoprecipitation. TmmllnQprecipitates ofthe viral particles with an
HFV specific chimpanzee serum or antiMuLV gag hybridoma supernatants were
CA 02229~1~ 1998-03-13
- 19 -
analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) along with their
corresponding cell lysates. HFV specific bands in immunoprccipitates from pelleted
virus or cellular Iysates were only observed in samples transfected with the HFV env
expression constructs, but not in samples expressing the MuLV amphotropic
5 envelope protein or mock transfected cultures. Two predominant HFV envelope
precursor bands of 130 and 110 KD were observed in immuno-precipitates of cellular
Iysates of HFV env transfected cells (12, 21). In addition, two bands corresponding
to the processed ~90 KD SU and the ~45-50 KD TM proteins could be observed
after longer exposure. The di~e-elll apparent sizes ofthe TM proteins in the cellular
10 samples transfected with the various HFV mllt~nts reflected the modifications in the
TM portion of the individual proteins. Only moderate differences in the steady state
level of the di~el el1t envelope proteins in the transfected cells were observed, except
for the HFV SSSMuLVR- and the HFV SSSMuLV proteins which showed a clearly
reduced cellular expression. Both envelope precursor proteins as well as the
15 processed SU and TM proteins were also detected in immll~oprecipitates of pelleted
viral particles. However, in general the relative ratio of processed proteins toprecursor proteins was increased in the viral particle immunoprecipitates compared
to the cell Iysates.
Interestingly, a good correlation between the amount of processed SU and
20 TM proteins in the individual immunoprecipitates of the viral particles and the
relative infectivity of the corresponding pseudotyped particles (Fig. 2) could be
observed. The HFV ~2MuLV chimeric envelope, which gave rise to pseudotyped
particles with the highest relative infectivity also showed the strongest SU and TM
bands in the RIPA. The amount of MuLV gag/pol proteins in the individual viral
25 particle preparations, as deterrmined in crude viral pellets or immunoplecip;~tes with
anti-gag hybridoma supernatants, was similar for all samples, except for the ~FV~2MuLV envelope transfection. This sample showed a significant decrease in
particle associated gag/pol proteins, indicating that fewer MuLV particles were
present in this p~e~alaLion compared to the other viral pellets. As a result, the
- CA 02229~1~ 1998-03-13
- - 20 -
relative amount of processed ~V SU and TM proteins per individual viral particlemay be even higher than estimated f'rom the immunoprecipitates of viral particles
preparations with HFV specific antibodies. A possible explanation for this
phenomenon is an enhanced absorbance of these particles by transfected cells notS expressing the HFV env protein, as a result of the increased infectivity of HFV
~2MuLV pseudotyped particles cornpared to particles pseudotyped by the other
HFV envelope proteins. This may result in a clearing of the HFV ~2MuLV
pseudotyped particles from the supernatant. Furthermore, in contrast to MuLV
particles pseudotyped with amphotropic MuLV envelope or VSV-G, HFV
pseudotyped viruses showed no reduction in infectivity in the absence of polycations
such as polybrene (30). Therefore, the relative titers of retroviral vectors
pseudotyped with HFV envelope by transient transfection may be underestimated
compared to pseudotypes with arnphotropic MuLV envelope or VSV-G. Further
experiments, however, using cell lines stably e,~ essillg the HFV envelope, which
should be resistant to infection by viruses pseudotyped with the HFV envelope, are
necessary to clarify these phenomena in more detail.
~. Inactivation of the splice donor and acceptor sites located into the FV env
gene
Furthermore, Bel-1 and Bet transcripts derived from intemal HFV promotor
(Pos. ~419 relative to the transcription start in the 5~ LTR), located within the HFV
envelope ORF (Pos. 6310-9276) efficiently utili~e a splice donor (SD, Pos. 9119
and a splice acceptor (SA, Pos. 9237) site within the coding region of TM subunit
of the env protein. Alte~ native splicing of mRNA coding for the HFV env proteinutili7ing these SD and SA sites results in potential envelope/bel fusion proteins. A
~170 KD can be detected in HFV infected cells by immunoprecipitation and the
mRNA is detectable by RT (reverse-transcriptase) PCR of total mRNA from HFV
infected human fibroblasts. Inactivation of the SD (Pos. 9119) by a GT->GG
mutation results in a disapearance of the 170 KD envelope fusion protein, while the
CA 02229~1~ 1998-03-13
- 21 -
expression ofthe 130 KD envelope precursor protein is not changed. The biological
function of the env/bel fusion proteins as well as the influence on viral titers of
pseudotyped MuLV particles are currently not known.
In summary, a system has been generated to produce MuLV based retroviral
5 vectors pseudotyped with HFV envelope proteins. The cytoplasmic domain of the
HFV envelope protein was at least partially involved in pseudotyping of MuLV
particles as progressive deletion of the cytoplasmic domain lead to a reduction in
gene transfer and incorporation of HFV env SU and TM subunits into the viral
particle. Addition of an unprocessed MuLV envelope cytoplasmic domain to one
deletion mutant, the HFV ~2MuLV envelope, resulted in a 10-20 fold increase in
infectivity compared to HFV wildtype envelope protein and an enhanced
incorporation of the chimeric envelope protein into pseudotyped particles. Retroviral
titers were 8-35 fold higher than those achieved by pseudotyping with the wildtype
HFV envelope protein. On some target cell types, the gene transfer efficiency was
15 similar or higher than those of retroviral vectors pseudotyped with the VSV-Gprotein. In the case of the wildtype MuLV envelope protein, the role of the
cytoplasmic domain for the specific incorporation of the envelope into the viralparticle is unclear. Some cytoplasmic tail deletion m~lt~ts resulted in a loss of
particle associated envelope proteins (15), whereas other mutant envelope proteins
20 showed little to no reduction in particle association (25~ 26). Our results argue for
a role of the MuLV env cytoplasmic domain in the particle association of the
envelope protein, at least in the enhanced incorporation of chimeric envelope
proteins into MuLV particles.
Recently, the pseudotyping of MuLV based retroviral vectors with foreign
25 envelope proteins, such as the VSV glycoprotein G (6, 38) or the GALV envelope
(2, 34), has resulted in an increase in virus stability, a broadened host-cell range and
an enhanced transduction efficiency of certain cell types. The broad host range of
FVs, the resistance to inactivation by human serum (30), and the efficient infection
of cells of various origin in the absence of polycations (unpublished observations and
CA 02229~1~ 1998-03-13
- 22 -
(30)) should make MuLV based retroviral vectors pseudotyped with the HFV
~2MuLV chimeric envelope protein a useful new tool for efficient gene transfer into
di~el ellL cell types. Unlike the expression of VSV-G, which is highly toxic for the
producer cells and has prevented the generation of stable VSV-G packaging cell
lines until recently (8, 22, 37), transient expression of the HFV ~2MuLV envelope
resulted in no apparent toxicity in 2g3T cells (data not shown, (19)).
EXAMPLE 2: ~IFV/SIV env chimeras
This example describes the construction of chimeric envelopes betweeen
HFV and SIV envelope proteins. Three different types of chimeric envelopes were
constructed. the sequence of the dirrelenl elements used herein after are available
in Genbank: SIV mm251 genome under accession number M19499, CMV
promoter under X03922 and pEGFP-CI (GFP gene) under U55763.
In the first type, the cytoplasmic tail of SIV is fused to the extracellular
domain (also designated in the literature ectodomain) of HFV within the
transmembrane anchor domain (constructs 1, 2 and 7 of Figure 4 and Figure SA).
The transmembrane anchor domain of the H~V env is deduced from its amino acid
sequence, and is therefore hypothetical. The transmembrane anchor domain of the
20 SIV env protein contains a small region of identical amino acids with respect to
HFV env. It is in this region that the fusion has been made. Thus the entire
extracellular domain of the chimeric env gene is from HFV, including the entire
surface subunit (SU) and most ofthe tr~n~memt-rane subunit (TM). The cytoplasrnic
tail is entirely from SIV.
In the second type of chimeric envelopes, the fusion genes is at the level of
the cleavage site between the SU and the TM subunits (constructs 3, 4 and 8 of
Figure 4 and Figure SB). Thus the SU subunit is from HFV and the TM subunit
from SIV. In the chirrleric envelope proteinS the cleavage site of the SIV env (Arg-
Gln-Lys-Arg) was used.
CA 02229~1~ 1998-03-13
- 23 -
In the third type of chimeric envelope, the SIV cytoplasmic tail is fused to
the HFV envelope in the cytoplasmic domain (constructs 5, 6 and 9 of Figure 4 and
Figure 5C). This construct is similar in design to the chimeric envelope ~2MuLV
and retains the first 6 cytoplasmic amino acids from the HFV envelope. The entire
5 cytoplasmic tail from SIV is fused to this envelope protein.
There exists two versions of the SIV envelope gene (see Figure 5D and
reference 42): a long form having a tail of 164 amino acids which is present in SIV
particles replicating in the natural host, the rhesus monkey (Macaca mulatta) and a
short form nammed ~hllm~ni7ed~ cont~ining only 18 amino acids. This short form
10 is selected for when virus isolated from the monkey are cultured on human cell lines.
Chimeric envelopes have been constructed using both tails. Constructs 1, 3 and 5comprise the long version and the r~ m~inin~ constructs the short version.
The possible presence of cis-acting repressor sequences (CRS) in the SIV
envelope genes may retain the env messP.nger RNA in the nucleus of producer cells.
15 The inhibitory effect on expression of env genes can be overcome if these messenger
RNAs contain the rev responsive element (RRE) and if, at the same time, rev is
expressed. This is the case for the sec,ond type of chimeric envelope constructs since
the RRE is present in the TM subunit of SIV and rev will be expressed from a
plasmid cont~ining the SIV proviral genGme. However, the RRE is not present in
20 the other envelope constructs. Although the exact nature of the CRS sequences in
the SIV env gene is not known, it is possible that the 3' splice site of the second
tat/rev exon in the env gene constitutes a CRS. We therefore decided to construct
a third variant of the chimeric env constructs in which this 3' splice site was
destroyed (43, 44, Figure 6). Destruction of the 3' splice site was only done in25 chimeric envelopes with the short, ~hnm~ni7eA)) cytoplasmic tail from SIV env.
In total 9 chimeric envelopes have been constructed (Figure 4~:
Env 1, 2 and 7 where fusion is in the trans membrane anchor domain, Env
1 has a long SIV cytoplasmic tail, Env 2 and 7 a short one. The 3' splice site in the
env gene is present in env 2 but has been deleted in env 7. The amino acid sequence
CA 02229~1~ 1998-03-13
- 24 -
of Env 2 and 7 are identical.
Env 3, 4 and ~ where fusion is at the cleavage site, Env 3 has a long
cytoplasmic tail, Env 4 and 8 a short one. The 3' splice site in the env gene is present
in env 4 but has been deleted in env 8. The amino acid sequence of Env 4 and 8 are
5 identical.
Env 5, 6 and 9 where fusion is in the cytoplasmic tail, Env 5 has a long
cytoplasmic tail, Env 6 and 9 a short one. The 3' splice site in the env gene is present
in env 6 but has been deleted in env 9. The amino acid sequence of Env 6 and 9 are
identical.
1. Construction of the chimeric env genes
All chimeric env genes have been cloned by replacing the appropriate HFV
env sequences in plasmid pczHFVenv wt with sequences from the SIV env gene.
pczHFVenv wt contains the HFV ~,vild type env encoding sequences cloned in
15 pcDNA3. 1/Zeo (Invitrogene) under the control of the CMV promoter. SIV env
sequences were amplified from a plasmid colllain.l1g the long version of the env gene
(pTG 664) for envelope constructs 1, 3 and 5 and from a plasmid cont~ining the
short version ofthe env gene (pTG 626Sma+) for the remaining constructs. These
two plasmids may be generated by a man skilled in the art by cloning SIV env
20 versions into p poly III*I (45). Amplification was carried out with an upstream
primer having a 5' extension of 50 nucleotides from the HFV env gene, and a
downstream primer having a 3' extension of 15 nucleotides conS~ining recognitionsites for the restriction enzymes Xhol and EcoKI. Both primers have a region of 20
nucleotides complementary to the SIV env gene permitting amplification of specific
25 parts of the SIV env genes.
After PCR of C-terminal parts of the SIVenv gene, amplimers were isolated
from gel and digested with Xho I to remove the 3' terminal nucleotides from the
amplimer. Plasmid pczHFVenv wt was digested with XhoI and EcoRI. The
amplimers were ligated to the linear pczHFVenv wt plasmid using the XhoI
~ CA 02229~1~ 1998-03-13
- - 25 -
redlln~nt ends. This results in a linear DNA fragment cont~ining the entire HFV
env gene and the cytoplasmic tail of SIV. The 5' end of the cytoplasmic tail contains
sequences homologous to the HFV e nv gene that is recombined by transformation
of this plasmid in E. coli BJ 5183 cells (46) according to the technology described
S in (41). This results in a closed circuklr plasmid in which the 3' terminal part of HFV
env has been replaced by the 3' end of SIV env. Using different combinations of
primers, all chimeric envelopes have been constructed this way (see Table 2). In the
resulting plasmid, the chimera encoding sequences are placed under the control of
the immediate early CMV promoter.
Table 2
upstr. primer downst. primer
Construct (SEQ ID) (SEQ ID) template DNA
Env 1OTG 11854(N0: 1) OTG 11855(N0: 2) pTG664
Env2 OTG 11854(N0: 1) OTG 11856(N0: 3) pTG626Sma+
Env 3OTG 11857(N0: 4) OTG 11855(N0: 2) pTG 664
Env 4OTG 11857(N0: 4) OTG 11856(N0: 3) pTG 626Sma+
Env SOTG 11858(N0: 5) OTG 11855(N0: 2) pTG 664
Env 6OTG 11858(N0: 5) OTG 11856(N0: 3) pTG 626Sma+
Env 7OTG 11854(N0: 1) OTG 11866(N0: 6) pTG626Sma+
Env 8OTG 11857(N0: 4) OTG 11866(N0: 6) pTG 626Sma+
Env9 OTG 11858(N0: 5) OTG 11866~ ): 6) pTG626Sma+
2. Production of pseudotyped SIV particles and transduction of target cells.
Production of pseudotyped retroviral particles is achieved by the co-
CA 02229~l~ l998-03-l3
- 26 -
transfection of 293 cells with a the different plasmids e~luressing the envelopechimeras and a plasmid cont~ining the proviral SIV genome in which env gene is
non functional due to the insertion of an expression cassette consisting of the
immediate early CMV promoter and the gene coding for the Enhanced Green
Fluorescent Protein (GFP)(nt 613 tc 1330 of Genbank sequence U5~763).
The protocol is the following: 293 cells were plated in 10 cm petri dishes
at a density of 2 X 1 o6 per dish in DMEM medium complemented with 10 % fetal
calf serum, non-essenti~l amino acids, gentamycin and glutamine (complete
DMEM). The next day, the cells were transfected using the standard Calcium
10 Phosphate transfection technique with 25 ~g SIV proviral plasmid and 5 ~g
envelope plasmid (the 9 chimeric envelope expression plasmids or control plasmids:
empty eA~-ession plasmid; VSV-G protein expression plasmid or pczHFVenv WT).
The next day, medium was removed and cells were washed once in DMEM me~illm
complçmented with 5 % fetal calf serum, non-~ssenti~l amino acids, gentamycin and
15 glut~mine and then incub~ted in 6 rnl ofthe same medium. Virus containing medium
was harvested two days later and cleared by centrifugation (5 min. at 3500 rpm).Target cells (293 or HT 1080) which were seeded the previous day at a density of5 x 105 cells per well of a 6-well plate were transduced as follows: target cells were
washed with 1 ml of DMEM (no serum), and then covered with 300 ~1l DMEM. 300
20 ~li virus cont~ining supematant supplemented with 10 ~g protamine sulfate/ml was
added and cells were incubated for 2 hours at 37 ~C in a 5% CO2 atmosphere. After
addition of 3 ml complete DMEM incubation was continued for 3 days.
Producer and target cells were analysed by FACScan as follows: cells were
trypsinized and washed with PBS. Cells were fixed with 1 ml PBS/4 %
25 formaldehyde for 10 minutes at 4 ~C. A~er one more wash step, cells were
resuspended in 1 ml PBS and analysed with a Becton-Dickinson FACScan using
Cell-Quest so~.ware. Fluorescence was measured using the FITC filter. All producer
cells were transfected with high efficiency. Transduction of control target cells was
as expected: VSV-G pseudotyped particles are able to transduce 293 cells and HT
CA 02229~1~ 1998-03-13
1080 cells. Supernatants from transfected cells without envelope protein or particles
pseudotyped with HFV env were not able to transduce any target cells. Viral
particles produced by the constructs 6 and 9 are able to transduce 293 and HT 1080
cells, showing that their chimeric en~l~elope is functional.
CA 02229~1~ 1998-03-13
- 28 -
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CA 02229~1~ 1998-03-13
SEQUENCE LISTING
(1~ GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: TRANSGENE S.A.
(B) STREET: 11 rue de Molsheim
(C) CITY: Strasbourg
(E) COUNTRY: France
(F) POSTAL CODE (ZIP): 67000
(G) TELEPHONE: 03 88 27 9l. 00
(H) TELEFAX: 03 88 27 91 41
(ii) TITLE OF INVENTION: expression of a foamy virus envelop protein
(iii) NUMBER OF SEQUENCES: 6
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Tape
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (EPO)
~2) INFORMATION FOR SEQ ID NO: 1:
ti) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 70 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iii) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: oligonucleotide de synthese oTG11854
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
TGGAACTGCC TTTAGTCTCT TGGGATACTT AAAGCCTATC CTAATAGGAG TAGGAGTAAT 60
ACTGTTAAGA 70
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE C~RACTERISTICS:
CA 02229~l~ l998-03-l3
-34-
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iii) ANTI-SENSE: YES
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: oligonucleotide de synthese oTG11855
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
GCCCTCGAGG AATTCTCACA AGAGCGTGAG CTCAA 35
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
tiii) HYPOTHETICAL: NO
~iii) ANTI-SENSE: YES
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: ol:igonucleotide de synthese oTG11856
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
GCCCTCGAGG AATTCCTACT GGA~ATAAGA GGGTG 35
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 70 base pairs
(B) TYPE: nucleic acid
(C) STRAN~EDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iii) ANTI-SENSE: NO
CA 02229~1~ 1998-03-13
-35-
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: o]igonucleotide de synthese oTG11857
(xi) SEQUENCE DESCRIPTION: SEQ ]:D NO: 4:
CTCCTTCCTA TCCCAATGTT ACTAGGGAAC ATTATACTTC CTGTAATAAT AGAAATAAAA 60
GAGGGGTCTT 70
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 70 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic:)
(iii) HYPOTHETICAL: NO
(iii) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: oligonucleotide de synthese oTG11858
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
TAGGGGTCAT TCTCTTGGTT ATTCTTATAT TTAAAATTGT ATCCTGGATT AAGTTAAGGC 60
AGGGGTATAG 70
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iii) ANTI-SENSE: YES
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: oligonucleotide de synthese oTG11866
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
GCCCTCGAGG AATTCCTATT GGAAATAAGA GGGTG 35
