U.S. patent application number 10/027760 was filed with the patent office on 2003-01-16 for non-primate lentiviral vectors and packaging systems.
Invention is credited to Looney, David J., Poeschla, Eric M., Wong-Staal, Flossie.
Application Number | 20030012769 10/027760 |
Document ID | / |
Family ID | 25468901 |
Filed Date | 2003-01-16 |
United States Patent
Application |
20030012769 |
Kind Code |
A1 |
Poeschla, Eric M. ; et
al. |
January 16, 2003 |
Non-primate lentiviral vectors and packaging systems
Abstract
The invention provides non-primate lentiviral vectors, packaging
cells and packaging plasmids based, for example, on feline and
ungulate retroviruses. In particular, the packaging plasmids are
designed for expression in human cells (which are also used as
packaging cells). The vectors of the invention transduce human
cells, including difficult to target non-dividing cells of the
hematopoietic and nervous system, in vitro and in vivo. The vectors
are suitable for general gene transfer to these cells and for gene
therapy to treat conditions mediated by these non-dividing cells
including cancer and HIV infection.
Inventors: |
Poeschla, Eric M.; (San
Diego, CA) ; Looney, David J.; (Encinitas, CA)
; Wong-Staal, Flossie; (San Diego, CA) |
Correspondence
Address: |
MORRISON & FOERSTER LLP
3811 VALLEY CENTRE DRIVE
SUITE 500
SAN DIEGO
CA
92130-2332
US
|
Family ID: |
25468901 |
Appl. No.: |
10/027760 |
Filed: |
December 21, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10027760 |
Dec 21, 2001 |
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09837644 |
Apr 17, 2001 |
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09837644 |
Apr 17, 2001 |
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08936633 |
Sep 24, 1997 |
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Current U.S.
Class: |
424/93.2 ;
435/235.1; 435/456; 514/44R |
Current CPC
Class: |
C12N 15/86 20130101;
C12N 2740/15043 20130101; C12N 2840/20 20130101 |
Class at
Publication: |
424/93.2 ;
514/44; 435/456; 435/235.1 |
International
Class: |
A61K 048/00; C12N
015/867; C12N 007/00 |
Goverment Interests
[0001] This invention was made with government support awarded by
the Veteran's Administration and under Grant No. Ca 67394 and
A136612 awarded by the National Institutes of Health. The
Government has certain rights in this invention.
Claims
What is claimed is:
1. A lentivirus transfer vector comprising a 5' LTR and a 3' LTR,
each of which contains a U3 region, wherein a part or all of a
regulatory element of the U3 region of the 5' LTR is replaced by
another regulatory element, operable in a mammalian cell, which is
not endogenous to said lentivirus.
2. The transfer vector of claim 1, wherein one or more nucleotide
bases of the U3 region of the 3' LTR are deleted.
3. The transfer vector of claim 1, wherein said regulatory element
not endogenous to said lentivirus is a cytomegalovirus enhancer,
promoter or enhancer/promoter.
4. The transfer vector of claim 1, wherein said regulatory element
not endogenous to said lentivirus is a Rous sarcoma virus enhancer,
promoter or enhancer/promoter.
5. The transfer vector of claim 1, wherein said transfer vector
further comprises a heterologous gene.
6. The vector of claim 1, wherein said lentivirus is human
immunodeficiency virus (HIV).
7. The transfer vector of claim 2, wherein said U3 region deleted
is all of said U3 region except for a 5' terminal dinucleotide and
an att sequence.
8. The transfer vector of claim 7, wherein said U3 region deleted
includes a TATA box sequence.
9. The transfer vector of claim 6, wherein said HIV is HIV-1.
Description
BACKGROUND OF THE INVENTION
[0002] Gene therapy provides methods for combating chronic
infectious diseases (e.g., HIV infection), as well as
non-infectious diseases including cancer and some forms of
congenital defects such as enzyme deficiencies. Several approaches
for introducing nucleic acids into cells in vivo, ex vivo and in
vitro have been used. These include liposome based gene delivery
(Debs and Zhu (1993) WO 93/24640 and U.S. Pat. No. 5,641,662);
Mannino and Gould-Fogerite (1988) BioTechniques 6(7): 682-691; Rose
U.S. Pat. No. 5,279,833; Brigham (1991) WO 91/06309; and Felgner et
al. (1987) Proc. Natl. Acad. Sci. USA 84: 7413-7414) and adenoviral
vector mediated gene delivery, e.g., to treat cancer (see, e.g.,
Chen et al. (1994) Proc. Nat'l. Acad. Sci. USA 91: 3054-3057; Tong
et al. (1996) Gynecol. Oncol. 61: 175-179; Clayman et al. (1995)
Cancer Res. 5: 1-6; O'Malley et al. (1995) Cancer Res. 55:
1080-1085; Hwang et al. (1995) Am. J. Respir. Cell Mol. Biol. 13:
7-16; Haddada et al. (1995) Curr. Top. Microbiol. Immunol. 199 (Pt.
3): 297-306; Addison et al. (1995) Proc. Nat'l. Acad. Sci. USA 92:
8522-8526; Colak et al. (1995) Brain Res. 691: 76-82; Crystal
(1995) Science 270: 404-410; Elshami et al. (1996) Human Gene Ther.
7: 141-148; Vincent et al. (1996) J. Neurosurg. 85: 648-654).
Replication-defective retroviral vectors harboring a therapeutic
polynucleotide sequence as part of the retroviral genome have also
been used, particularly with regard to simple MuLV vectors. See,
e.g., Miller et al. (1990) Mol. Cell. Biol. 10:4239 (1990); Kolberg
(1992) J. NIH Res. 4:43, and Cornetta et al. Hum. Gene Ther. 2:215
(1991)).
[0003] One of the most attractive targets for gene therapy is HIV
infection. The pandemic spread of HIV has driven an intense
world-wide effort to unravel the molecular mechanisms and life
cycle of these viruses. It is now clear that the life cycle of HIVs
provide many potential targets for inhibition by gene therapy,
including cellular expression of transdominant mutant gag and env
nucleic acids to interfere with virus entry, TAR (the binding site
for tat, which is typically required for transactivation) decoys to
inhibit transcription and trans activation, and RRE (the binding
site for Rev; i.e., the Rev Response Element) decoys and
transdominant Rev mutants to inhibit RNA processing. See, Rosenburg
and Fauci (1993) in Fundamental Immunology, Third Edition Paul (ed)
Raven Press, Ltd., New York and the references therein for an
overview of HIV infection and the HIV life cycle. Gene therapy
vectors encoding ribozymes, antisense molecules, decoy genes,
transdominant genes and suicide genes, including retroviruses are
described in Yu et al., Gene Therapy (1994) 1:13-26. Antisense and
ribozyme therapeutic agents are of increasing importance in the
treatment and prevention of HIV infection.
[0004] Despite the various gene therapeutic approaches now underway
for treating cancer, HIV and the like, there are a variety of
limitations of the delivery systems currently used in gene therapy.
For instance, with regard to HIV treatment, the extensively used
murine retroviral vectors transduce (transfer nucleic acids into)
human peripheral blood lymphocytes poorly, and fail to transduce
non-dividing cells such as monocytes/macrophages, which are known
to be reservoirs for HIV. New safer vectors for the delivery of
viral inhibitors, particularly to non-dividing hematopoietic stem
cells for the treatment of HIV infection, are desirable.
[0005] Non-primate lentiviruses provide a possible system for the
development of new vector systems; however, relatively little is
known about these viruses. Although their biology has received
considerably less scrutiny than that of the primate lentiviruses
(e.g., HIV-1, HIV-2 and SIV), non-primate lentiviruses are of
interest for comparative lentivirus biology and as potential
sources of safer lentiviral vectors. HIV-based retroviral vectors
have recently shown promise for therapeutic gene transfer because
they display the lentiretrovirus-specific property of permanently
infecting non-dividing cells (see, Naldini et al. (1996) Science
272, 263-267). In contrast, retroviral vectors derived from simpler
retroviruses (e.g., the Oncovirinae) require breakdown of the
nuclear envelope during mitosis to complete reverse transcription
and integration. Consequently, these vectors transduce non-dividing
cells poorly, which may limit usefulness for gene transfer to
quiescent or post-mitotic cellular targets. However, HIV vectors
present complex safety problems (see, Emerman (1996) Nature
Biotechnology 14, 943).
[0006] The non-primate lentiviruses include the ungulate
lentiviruses, including visna/maedi virus, caprine arthritis
encephalitis virus (CAEV), equine infectious anemia virus (EIAV),
and bovine immunodeficiency virus (BIV). These lentiviruses only
infect hoofed animals (ungulates) and generally only infect
particular species of ungulates.
[0007] The non-primate lentiviruses also include feline
immunodeficiency virus (FIV) (see, Clements & Zink (1996)
Clinical Microbiology Reviews 9, 100-117), which only infects
felines. Numerous strains of FIV have been identified.
[0008] Non-primate (e.g., feline and ungulate) lentiviruses may
provide a safer alternative than primate lentiviral vectors, but
their use is complicated by a relative lack of knowledge about
their molecular properties, especially their adaptability to
non-host animal cells (Emerman, id). All lentiviruses display
highly restricted tropisms (see, Clements & Zink (1996), supra,
and Haase (1994) Annals of the New York Academy of Sciences 724,
75-86).
[0009] FIV was discovered in 1986 as a cause of acquired immune
deficiency and neurological disease in, and only in, domestic cats
(Felis catus) Pedersen et al. (1987) Science 235, 790-793 (1987);
Elder & Phillips (1993) Infectious Agents and Disease 2,
361-374; Pedersen (1993) "The feline immunodeficiency virus" in The
Retroviridae (ed. Levy, J. A.) 181-228 (Plenum Press, New York
Bendinelli et al. (1995) Clinical Microbiology Reviews 8, 87-112;
and, Sparger (1993) Veterinary Clinics of North America, Small
Animal Practice 23, 173-191). In the great cats, FIV is widely
dispersed geographically and appears to be commensal: 18 of 37
species of free-roaming, non-domestic Felidae are known to be
infected world-wide, but none develop disease (Elder & Phillips
(1993), supra; Olmsted et al. (1992) Journal of Virology 66,
6008-6018; Barr et al. (1995) Journal of Virology 69, 7371-7374;
Courchamp & Pontier (1994) "Feline immunodeficiency virus: an
epidemiological review." Comptes Rendus de L Academie des Sciences.
Serie III, Sciences de la Vie 317, 1123-1134). The virus is
prevalent, infecting 2-20% of domestic cat populations in North
America, Europe and Japan; higher rates are seen in cats brought to
veterinary attention (Pedersen (1993), supra and Courchamp, F.
& Pontier (1994), supra). The worldwide prevalence of FIV in
diverse Felidae and the observation that Felis catus sera dating to
the 1960's show similar high rates of positivity, suggest that FIV
has not been recently introduced into domestic cats (Bendinelli et
al. (1995), supra, Olmsted et al. (1992), supra; Courchamp, F.
& Pontier (1994) supra; Shelton et al. (1990) Journal of
Acquired Immune Deficiency Syndromes 3, 623-630; Bennett &
Smyth (1992) British Veterinary Journal 148, 399-412; Brown et al.
(1993) Journal of Zoo and Wildlife Medicine 24, 357-364; Carpenter
& O'Brien (1995) Current Opinion in Genetics and Development 5,
739-745.
[0010] There is no evidence for FIV infection of non-felids.
Cross-infection by any of the ungulate or feline lentiviruses has
never been observed in non-ungulates, or non-felids respectively.
HIV and FIV differ notably in their modes of transmission since FIV
is spread principally by biting (Pederson (1993), supra). Despite
frequent exposure of humans to FIV through bites by domestic cats,
this plausibly efficient means of inoculation does not result in
human seroconversion or any other detectable evidence of human
infection or disease (Pedersen (1993) id.; Bendinelli et al. (1995)
supra.; Sparger (1993), supra; Courchamp, F. & Pontier (1994),
supra; Brown et al. (1993).
[0011] FIV is also genetically and antigenically distant from the
primate lentiviruses. Nucleotide sequence comparisons indicate a
closer relationship to the ungulate lentiviruses than to HIV and
SIV (Olmsted et al. (1989) Proceedings of the National Academy of
Sciences of the United States of America 86, 2448-2452 (Olmsted et
al. (1989) A); Olmsted et al. (1989) Proceedings of the National
Academy of Sciences of the United States of America 86, 8088-8092
(Olmstead et al. (1989) B). Serological cross reactivity of FIV
core proteins to several ungulate lentiviruses has been observed
but none occurs to HIV-1, HIV-2 or SIV (Elder, J. H. & Phillips
(1993), supra; Bennett, M. & Smyth (1992), supra; Olmsted et
al. (1989) A, supra; Talbott et al. (1989) Proceedings of the
National Academy of Sciences of the United States of America 86,
5743-5747; Miyazawa et al. (1994) Archives of Virology 134,
221-234). The virus encodes a dUTPase; this fifth pol-encoded
enzymatic activity is a feature found only in non-primate
lentiviruses (Wagaman et al. (1993) Virology 196, 451-457).
Phylogenetic and epidemiologic data suggest an ancient adaptive
episode between FIV and ancestors of wild felines as well as early
evolutionary divergence from ancestors of other lentiviruses
(Olmsted, R. A. et al. (1992), supra; Brown et al. (1993), supra;
Carpenter & O'Brien (1995) Talbott et al. (1989), supra.).
[0012] At the cellular level, restrictions in human cells to both
the productive and infective stages of the non-primate lentivirus
life cycles are also evident (Tomonaga et al. (1994) Journal of
Veterinary Aledical Science 56, 199-201; Miyazawa et al. (1992)
Journal of General Virology 73, 1543-1546; Thormar &
Sigurdardottir (1962) Acta Pathol Microbiol Scandinav 55, 180-186;
Gilden et al. (1981) Archives of Virology 67, 181-185; Carey &
Dalziel (1993) British Veterinary Journal 149, 437-454. The bases
for these blocks, which might impede development of non-primate
lentivirus-based vectors for human application, are not well
understood.
[0013] The CD4 molecules of primates are the only known lentivirus
primary receptors (e.g., for HIV). Neither primary nor secondary
receptors have previously been determined for any of the
non-primate lentiviruses. Although an antibody that binds to the
feline homologue of CD9 inhibits FIV infection in tissue culture
(Willett (1994) Immunology 81, 228-233), subsequent investigation
has established that neither CD9 nor the feline homologue of CD4
are FIV receptors (Willett et al. (1997) Journal of General
Virology 78, 611-618). Reported obstacles to FIV expression in
human cells have included poor function of core viral functions
such as the Rev/RRE regulatory axis (Tomonaga, K. et al. (1994)
supra; Simon et al. (1995) Journal of Virology 69, 4166-4172) and
poor promoter activity of the long terminal repeat (LTR) (Miyazawa
et al. (1992), supra; Sparger et al. (1992) Virology 187, 165-177.
Because of these blocks, expression of the non-primate lentivirus
Rev-dependent structural proteins in non-host animal cells has
received very limited study.
[0014] In addition to the question of restricted tropism,
production of non-primate lentivirus vectors in ungulate or feline
cells for clinical use would create hazards for transmission of
endogenous retroviruses or other potential pathogens. This risk has
been documented for cells of diverse animal origin (Stoye &
Coffin (1995) Nature Medicine 1, 1100; van der Kuyl et al. (1997)
Journal of Virology 71, 3666-3676). Feline cells also contain
multiple copies of a replication-competent, type C endogenous
retrovirus (RD 114) that replicates in human cells, phenotypically
mixes with other retroviruses, and is related at the nucleotide
sequence level to a primate retrovirus (baboon endogenous virus)
(McAllister et al (1972) Nature New Biol 235, 3-6). Moreover,
unlike most other type C mammalian retroviruses, RD114 resists
inactivation by human serum complement (Takeuchi et al. (1994)
Journal of Virology 68, 8001-8007). Cat cells may also contain
other unknown and potentially pathogenic infectious agents.
[0015] Accordingly, there is a need in the art for safer lentiviral
vectors, e.g., for the delivery of genes, cancer therapeutics,
viral inhibitors and the like to non-dividing cells, including
hematopoietic stem cells and neuronal cells, and for human vector
packaging cells capable of packaging non-primate lentiviral
vectors. The present invention provides these and other
features.
SUMMARY OF THE INVENTION
[0016] This invention describes retroviral packaging systems,
vectors, packagable nucleic acids and other features based on the
discovery and design of non-primate lentiviral vectors which are
active in human cells. The vectors, which are packageable by a
non-primate lentivirus such as FIV, transduce non-dividing human
cells. The packaging systems produce vector packaging components in
trans in human cells, thereby avoiding the possibility of
introducing new pathogens into the human population. These vectors
and packaging components are useful for construction of general
gene transfer vectors and for human gene therapy.
[0017] One class of retroviral vector provided by the invention has
a vector nucleic acid packaged by a non-primate letivirus, such as
FIV or an ungulate retrovirus. Thus the vector has a packagable
nucleic acid which is recognized and packaged by the viral
packaging proteins encoded by the selected retrovirus (e.g., FIV).
The vector nucleic acid also includes a heterologous target nucleic
acid such as a therapeutic gene. The vector nucleic acid is not
virulent, because the nucleic acid lacks, or is defective, for one
or more gene necessary for viral replication. However, when the
missing or defective component (e.g., a retroviral protein) is
provided in trans (e.g., in a packaging cell) the vector nucleic
acid is packaged in a retroviral viral particle. The vectors
optionally include vector packaging or replication elements such as
viral proteins, viral particles, reverse transcriptase activity, or
the like.
[0018] One preferred class of targets for the vectors of the
invention are human cells, particularly non-dividing cells such as
terminally differentiated hematopoietic cells and neurons (the
cells are in vitro or in vivo). Accordingly, features directed to
transduction and infection of human cells are preferred features.
For example, incorporation of vesicular stomatitis virus (VSV)
glycoprotein on the surface of the vector is preferred in some
embodiments, as this facilitates entry of the vector into a variety
of human cells. Similarly, incorporation of a promoter (e.g., the
CMV promoter or a t-RNA promoter) which directs expression of one
or more nucleic acid encoded by the vector in a human cell is
desirable for production of nucleic acids and, optionally, proteins
encoded by the vector in a human target cell.
[0019] Packaging plasmids which encode viral components which
package the vector nucleic acids in trans are also provided. The
packaging plasmids include a promoter which is active in a human
cell (i.e., a human cell used for vector packaging). This promoter
is operably linked to a nucleic acid encoding at least one protein
necessary for packaging the vector nucleic acid, e.g., an FIV or
other non-primate lentivirus packaging nucleic acid. The packaging
plasmid lacks an FIV packaging site.
[0020] In one preferred embodiment, the packaging plasmid has an
FIV LTR having a U3 promoter deletion, typically with a
heterologous promoter insertion into the deletion site. This
arrangement results in eliminating endogenous FIV LTR promoter
function and permitting regulation by the heterologous
promoter.
[0021] It will be appreciated that retroviral packaging cells which
package non-primate lentiviral packagable nucleic acids are
provided by the packaging plasmids described above. In particular,
the cells, which are preferably human, comprise packaging plasmids
encoding necessary viral packaging elements (e.g., Gag and Env
proteins). These packaging elements are used to package packageable
vector nucleic acids in trans. For safety reasons, it is often
preferable for the cell to include separate packaging plasmids,
each of which encode different packaging proteins. For example, a
packaging cell can include two separate packaging plasmids encoding
distinct retroviral packaging proteins (e.g., FIV Gag and Env
proteins). The cell can further comprise a plasmid which encodes a
pseudotyping element such as the VSV envelope glycoprotein to
expand the range of any packaged plasmid. The psudotyping element
can be encoded on the same plasmid as other non-primate retroviral
elements, or on a separate plasmid. In any case, desired packaging
elements are under the control of suitable regulatory elements
which direct expression of the components in a human cell. It will
be appreciated that packageble nucleic acids (e.g., which include
an FIV packaging site and a heterologous nucleic acid) are
optionally transduced (stably or transiently) into the cells of the
invention.
BRIEF DESCRIPTION OF THE DRAWING
[0022] FIG. 1 is a schematic describing nucleic acid constructs of
the invention, including FIV34TF-10, CT5, CTRZLb and CTAGCb.
[0023] FIG. 2 panels A and B provide experimental results is
graphic form.
DEFINITIONS
[0024] For purposes of the present invention, the following terms
are defined below.
[0025] A "vector" is a composition which can transduce, transform
or infect a cell, thereby causing the cell to express vector
encoded nucleic acids and, optionally, proteins other than those
native to the cell, or in a manner not native to the cell. A vector
includes a nucleic acid (ordinarily RNA or DNA) to be expressed by
the cell (a "vector nucleic acid"). A vector optionally includes
materials to aid in achieving entry of the nucleic acid into the
cell, such as a retroviral particle, liposome, protein coating or
the like.
[0026] A "non-primate lentivirus packageable nucleic acid" is a
nucleic acid having a functional virus packaging site from an
ungulate lentivirus or FIV lentivirus. Nucleic acids having this
packaging site which can be incorporated into a viral particle by
viral components supplied in trans by a corresponding wild-type
virus are packaged by the wild-type virus (or appropriate packaging
components derived from a wild-type virus).
[0027] A "packaging defect which blocks self packaging" of a
non-primate lentiviral vector nucleic acid is an inability of the
nucleic acid to produce at least one viral protein necessary for
packaging the vector nucleic acid into a viral particle in the
context of a cell. For example, when Gag or Env proteins are not
encoded by the lentiviral vector, the proteins must be supplied in
trans before the vector nucleic acid can be packaged in the cell.
The omission can be a deletion or mutation of a gene necessary for
viral packaging from a viral clone, in the coding or non-coding
(e.g., promoter) region of the relevant gene. The vector nucleic
acid is trans-rescuable when it encodes a viral packaging site
which is recognized be a non-primate lentiviral vector such as
FIV.
[0028] A "packaging plasmid" is a plasmid which encodes components
necessary for production of viral particles by a cell transduced by
the packaging plasmid. The packaging plasmid optionally encodes all
of the components necessary for production of viral particles, or
optionally includes a subset of the components necessary for
packaging. For instance, in one preferred embodiment, a packaging
cell is transformed with more than one packaging plasmid, each of
which has a complementary role in the production of a non-primate
lentiviral particle (e.g., for FIV). The packaging plasmid lacks a
functional packaging site, i.e., a packaging site recognized by the
non-primate lentivirus components encoded by the plasmid, rendering
the plasmid incapable of self packaging.
[0029] Two (or more) non-primate lentivirus based packaging
plasmids are "complementary" when they together encode all of the
functions necessary for viral packaging, and when each individually
does not encode all of the functions necessary for packaging. Thus,
e.g., when two plasmids transduce a single cell and together they
encode the information for production of FIV packaging particles,
the two vectors are "complementary." The use of complementary
plasmids is preferred because it increases the safety of any
packaging cell made by transformation with a packaging plasmid by
reducing the possibility that a recombination event will produce an
infective virus.
[0030] Packaging plasmids encode components of a viral particle.
The particles are competent to package target RNA which has a
corresponding packaging site. "High efficiency packaging plasmids"
package target RNAs having packaging sites such that packaging
cells transiently or stably transduced with the packaging plasmid
and transduced with a target packageable nucleic acid target
packageable vector RNA at a titer of at least about 105 or 106 to
about 107 or even 108 transducing units per ml or more. The precise
titer which is produced varies depending on the nature of the
packageable nucleic acid and the packaging cell selected. Higher
infectivities are typically obtained when packaging vectors with
complete packaging sites.
[0031] An "inhibitor" or "viral inhibitor" is most typically a
nucleic acid which encodes an active anti-viral agent, or is itself
an anti-viral agent. Thus, in one class of embodiments, the
inhibitor is a "direct inhibitor," i.e., the inhibitor acts
directly on a viral component to inhibit the infection,
replication, integration or growth of the virus in the cell. For
instance, in one particularly preferred embodiment, the inhibitor
comprises a trans-active ribozyme which cleaves an HIV transcript.
In this configuration, the inhibitor is typically an RNA molecule
with catalytic nuclease activity. In another class of embodiments,
the inhibitor is an "indirect inhibitor," i.e., the inhibitor
encodes the direct inhibitor. An inhibitor "encodes" a direct
inhibitor such as an active ribozyme, protein, RNA molecular decoy,
or anti-sense RNA if it contains either the sense or anti-sense
coding or complementary nucleic acid which corresponds to the
direct inhibitor. By convention, direct inhibitor RNAs such as
ribozymes are typically listed as their corresponding DNA
sequences. This is done to simplify visualization of the
corresponding active RNA, which is equivalent to the given sequence
with the T residues replaced by U residues.
[0032] "Viral inhibition" refers to the ability of a component to
inhibit the infection, growth, integration, or replication of a
virus in a cell. Inhibition is typically measured by monitoring
changes in a cell's viral load (i.e., the number of viruses and/or
viral proteins or nucleic acids present in the cell, cell culture,
or organism) or by monitoring resistance by a cell, cell culture,
or organism to infection.
[0033] A "promoter" is an array of nucleic acid control sequences
which direct transcription of a nucleic acid. As used herein, a
promoter includes necessary nucleic acid sequences near the start
site of transcription, such as, in the case of a polymerase II type
promoter, a TATA element. A promoter also optionally includes
distal enhancer or repressor elements which can be located as much
as several thousand base pairs from the start site of
transcription.
[0034] A "constitutive" promoter is a promoter which is active
under most environmental and developmental conditions. An
"inducible" promoter is a promoter which is under environmental or
developmental regulation.
[0035] The terms "isolated" or "biologically pure" refer to
material which is substantially or essentially free from components
which normally accompany it as found in its native state.
[0036] The term "heterologous" when used with reference to portions
of a nucleic acid indicates that the nucleic acid comprises two or
more subsequences which are not found in the same relationship to
each other in nature. For instance, the nucleic acid is typically
recombinantly produced, having two or more sequences from unrelated
genes arranged to make a new functional nucleic acid. For example,
in one embodiment, the nucleic acid has a promoter from one gene
arranged to direct the expression of a coding sequence from a
different gene, such as an anti-viral ribozyme. Thus, with
reference to the ribozyme coding sequence, the promoter is
heterologous.
[0037] The term "identical" in the context of two nucleic acid or
polypeptide sequences refers to the residues in the two sequences
which are the same when aligned for maximum correspondence. When
percentage of sequence identity is used in reference to proteins or
peptides it is recognized that residue positions which are not
identical often differ by conservative amino acid substitutions,
where amino acids residues are substituted for other amino acid
residues with similar chemical properties (e.g. charge or
hydrophobicity) and therefore do not change the functional
properties of the molecule. Where sequences differ in conservative
substitutions, the percent sequence identity may be adjusted
upwards to correct for the conservative nature of the substitution.
Means for making this adjustment are well known to those of skill
in the art. Typically this involves scoring a conservative
substitution as a partial rather than a full mismatch, thereby
increasing the percentage sequence identity. Thus, for example,
where an identical amino acid is given a score of 1 and a
non-conservative substitution is given a score of zero, a
conservative substitution is given a score between zero and 1. The
scoring of conservative substitutions is calculated, e.g.,
according to the algorithm of Meyers and Miller, Computer Applic.
Biol. Sci., 4: 11-17 (1988) e.g., as implemented in the program
PC/GENE (Intelligenetics, Mountain View, Calif., USA). A
"comparison window", as used herein, refers to a segment of at
least about 50 contiguous positions, usually about 50 to about 200,
more usually about 100 to about 150 in which a sequence is compared
to a reference sequence of the same number of contiguous positions
after the two sequences are optimally aligned. Methods of alignment
of sequences for comparison are well known in the art. Optimal
alignment of sequences for comparison may be conducted by the local
homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2:
482; by the homology alignment algorithm of Needleman and Wunsch
(1970) J. Mol. Biol. 48: 443; by the search for similarity method
of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444;
by computerized implementations of these algorithms (including, but
not limited to CLUSTAL in the PC/Gene program by Intelligenetics,
Mountain View, Calif., GAP, BESTFIT, FASTA, and TFASTA in the
Wisconsin Genetics Software Package, Genetics Computer Group (GCG),
575 Science Dr., Madison, Wis., USA); the CLUSTAL program is well
described by Higgins and Sharp (1988) Gene, 73: 237-244 and Higgins
and Sharp (1989) CABIOS 5: 151-153; Corpet, et al. (1988) Nucleic
Acids Research 16, 10881-90; Huang, et al. (1992) Computer
Applications in the Biosciences 8, 155-65, and Pearson, et al.
(1994) Methods in Molecular Biology 24, 307-31. Alignment is also
often performed by inspection and manual alignment. "Conservatively
modified variations" of a particular nucleic acid sequence refers
to those nucleic acids which encode identical or essentially
identical amino acid sequences, or where the nucleic acid does not
encode an amino acid sequence, to essentially identical sequences.
Because of the degeneracy of the genetic code, a large number of
functionally identical nucleic acids encode any given polypeptide.
For instance, the codons CGU, CGC, CGA, CGG, AGA, and AGG all
encode the amino acid arginine. Thus, at every position where an
arginine is specified by a codon, the codon can be altered to any
of the corresponding codons described without altering the encoded
polypeptide. Such nucleic acid variations are "silent variations,"
which are one species of "conservatively modified variations."
Every nucleic acid sequence herein which encodes a polypeptide also
describes every possible silent variation. One of skill will
recognize that each codon in a nucleic acid (except AUG, which is
ordinarily the only codon for methionine) can be modified to yield
a functionally identical molecule by standard techniques.
Accordingly, each "silent variation" of a nucleic acid which
encodes a polypeptide is implicit in any described sequence.
Furthermore, one of skill will recognize that individual
substitutions, deletions or additions which alter, add or delete a
single amino acid or a small percentage of amino acids (typically
less than 5%, more typically less than 1%) in an encoded sequence
are "conservatively modified variations" where the alterations
result in the substitution of an amino acid with a chemically
similar amino acid. Conservative substitution tables providing
functionally similar amino acids are well known in the art. The
following six groups each contain amino acids that are conservative
substitutions for one another: 1) Alanine (A), Serine (S),
Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3)
Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5)
Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6)
Phenylalanine (F), Tyrosine (Y), Tryptophan (W). See also,
Creighton (1984) Proteins W. H. Freeman and Company. Finally, the
addition of sequences which do not alter the activity of a nucleic
acid molecule, such as a non-functional sequence is a conservative
modification of the basic nucleic acid.
[0038] "Stringent hybridization wash conditions" in the context of
nucleic acid hybridization experiments such as Southern and
northern hybridizations are sequence dependent, and are different
under different environmental parameters. An extensive guide to the
hybridization of nucleic acids is found in Tijssen (1993)
Laboratory Techniques in Biochemistry and Molecular
Biology--Hybridization with Nucleic Acid Probes part I chapter 2
"overview of principles of hybridization and the strategy of
nucleic acid probe assays", Elsevier, New York. Generally, highly
stringent hybridization and wash conditions are selected to be
about 5.degree. C. lower than the thermal melting point (T.sub.m)
for the specific sequence at a defined ionic strength and ph. The
T.sub.m is the temperature (under defined ionic strength and pH) at
which 50% of the target sequence hybridizes to a perfectly matched
probe. Very stringent conditions are selected to be equal to the
T.sub.m for a particular probe.
[0039] An example of stringent hybridization conditions for
hybridization of complementary nucleic acids which have more than
100 complementary residues on a filter in a Southern or northern
blot is 50% formalin with 1 mg of heparin at 42.degree. C., with
the hybridization being carried out overnight. An example of
stringent wash conditions is a 0.2.times. SSC wash at 65.degree. C.
for 15 minutes (see, Sambrook, supra for a description of SSC
buffer). Often the high stringency wash is preceded by a low
stringency wash to remove background probe signal. An example low
stringency wash is 2.times. SSC at 40.degree. C. for 15 minutes. In
general, a signal to noise ratio of 2.times. (or higher) than that
observed for an unrelated probe in the particular hybridization
assay indicates detection of a specific hybridization.
[0040] Nucleic acids which do not hybridize to each other under
stringent conditions are still substantially identical if the
polypeptides which they encode are substantially identical. This
occurs, e.g., when a copy of a nucleic acid is created using the
maximum codon degeneracy permitted by the genetic code.
[0041] An "FIV-1 Gag protein" is a protein encoded by the FIV-1 gag
gene. The Gag proteins are typically translated as a large
preprotein which is cleaved to form the structural core proteins
which package wild-type FIV genomic RNA. A truncated Gag protein is
a protein produced from a gag gene having a deletion relative to
the wild-type sequence. Similarly, an FIV reverse transcriptase
protein and an FIV envelope protein are encoded by the pol and env
genes, respectively.
[0042] The term "nucleic acid" refers to a deoxyribonucleotide or
ribonucleotide polymer in either single- or double-stranded form,
and unless otherwise limited, encompasses known analogues of
natural nucleotides that hybridize to nucleic acids in manner
similar to naturally occurring nucleotides. Unless otherwise
indicated, a particular nucleic acid sequence optionally includes
the complementary sequence thereof.
[0043] The term "operably linked" refers to a functional linkage
between a nucleic acid expression control sequence (such as a
promoter, or array of transcription factor binding sites) and a
second nucleic acid sequence, wherein the expression control
sequence directs transcription of the nucleic acid corresponding to
the second sequence.
[0044] The term "recombinant" when used with reference to a cell
indicates that the cell replicates or expresses a nucleic acid, or
expresses a peptide or protein encoded by nucleic acid whose origin
is exogenous to the cell. Recombinant cells can express genes that
are not found within the native (non-recombinant) form of the cell.
Recombinant cells can also express genes found in the native form
of the cell wherein the genes are re-introduced into the cell by
artificial means.
[0045] A "recombinant expression cassette" or simply an "expression
cassette" is a nucleic acid construct, generated recombinantly or
synthetically, with nucleic acid elements which permit
transcription of a particular nucleic acid in a cell. The
recombinant expression cassette can be part of a plasmid, virus, or
nucleic acid fragment. Typically, the recombinant expression
cassette includes a nucleic acid to be transcribed, and a promoter.
In some embodiments, the expression cassette also includes, e.g.,
an origin of replication, and/or chromosome integration elements
(e.g., a retroviral LTR).
[0046] The term "subsequence" in the context of a particular
nucleic acid sequence refers to a region of the nucleic acid equal
to or smaller than the specified nucleic acid.
[0047] A virus or vector "transduces" a cell when it transfers
nucleic acid into the cell. A virus or vector is "infective" when
it transduces a cell, replicates, and (without the benefit of any
complementary virus or vector) spreads progeny vectors or viruses
of the same type as the original transducing virus or vector to
other cells in an organism or cell culture, wherein the progeny
vectors or viruses have the same ability to reproduce and spread
throughout the organism or cell culture. Thus, for example, a
nucleic acid encoding an FIV particle is not infective if the
nucleic acid cannot be packaged by the FIV particle (e.g., if the
nucleic acid lacks an FIV packaging site), even though the nucleic
acid can be used to transfect and transform a cell. Similarly, an
FIV-packageable nucleic acid packaged by an FIV particle is not
infective if it does not encode the FIV particle that it is
packaged in, even though it may be used to transform and transfect
a cell. Vectors which do not encode a complete set of viral
packaging components (e.g., Gag and Env proteins) are "packaging
deficient." These vectors are "transrescuable" when the vectors are
packaged by viral proteins supplied in trans in a packaging cell.
If an FIV-packageable nucleic acid is used to transform a cell
infected with FIV in a cell culture or organism infected with FIV,
the FIV-packageable nucleic acid will be replicated and
disseminated throughout the organism in concert with the infecting
FIV virus. However, the FIV-packageable nucleic acid is not itself
"infective", because packaging functions are supplied by the
infective FIV virus via trans complementation.
[0048] A cell "supernatant" is the culture medium in which a cell
is grown. The culture medium includes material from the cell,
including, e.g., FIV viral particles which bud off from the cell
membrane and enter the culture medium.
DETAILED DISCUSSION OF THE INVENTION
[0049] Transfection of the non-primate lentiviral clone CF1 into
human HeLa, 293 and 293-T kidney cells by the calcium phosphate
co-precipitation method produced the surprising result of
widespread ballooning syncytia (multi-nucleated giant cells
produced by fusion of envelope-expressing cells with other cells)
containing fifty to several hundred nuclei and high levels (over
one million cpm) of supernatant reverse transcriptase.
Unexpectedly, human 293 and 293-T cell and HeLa monolayer cultures
were reproducibly 90-95% destroyed by syncytia after transient
transfection of CF1 (for example by transfection of 10 yg of CF1 in
a 75 cm.sup.2 flask), yet, no infectious or replication-competent
virus was produced: transfer of large volumes of CF1-transfected
293-T cell supernatant to fresh 293 or 293-T cells or to Crandall
feline kidney cells resulted in no syncytia or RT production.
CF1.DELTA.env, which is identical to CF1 except for an FIV envelope
deletion does not cause syncytia but does produce high levels of
reverse transcriptase and of viral functions needed for packaging
of vectors. These unprecedented results produced the novel insight
that all the functions of non-primate vectors such as FIV needed
for protein production, including the Rev/RRE axis of regulation,
FIV gag/pol production and envelope-mediated syncytia, could take
place in human cells if the FIV promoter were replaced with a
promoter active in human cells.
[0050] Accordingly, this invention provides the first use of
non-primate lentiviral vectors packaged in human packaging cells
for transduction of human target cells. Prior to the present
invention there was no way of knowing whether these packaging
systems could be developed in human cells, or whether packaged
non-primate lentiviral vectors could be used to transduce human
cells, or whether the regulatory, processing and packaging
components of such vectors would be active in human cells. There
was no information on desirable arrangements or particular
constructs especially useful in the vectors and packaging cells of
the invention, e.g., the arrangement and construction of FIV based
vectors and packaging systems described herein. Advantages to the
vectors include a lack of human pathogenicity for the viruses that
the vectors and packaging systems are based upon and the ability of
the vectors to transduce non-dividing cells human cells. Such
non-dividing cells include, but are not limited to, cells of the
human nervous system, eye, hematopoietic system, integument,
endocrine system, hepatobiliary system, gastrointestinal tract,
genitourinary tract, bone, muscle, cardiovascular system and
respiratory system. All of these cells are transduced in vitro or
in vivo using the vectors of the invention. These vectors also
prevent exposure of patients to non-human cells and prevent
exposure to lentiviral genes or lentiviral proteins derived from
known pathogens.
[0051] The mechanisms of the FIV life cycle in human cells are
described herein. It is reported here for the first time that
Feline immunodeficiency virus (FIV) proteins encoded by a packaging
plasmid can be expressed at high levels in human cells, in
replication-defective fashion, and supplied to FIV packagable
vectors in trans, by replacing the long terminal repeat of the FIV
genome with a heterologous promoter such as the human
cytomegalovirus immediate early promoter (CMV promoter). In
particular, substitution of a heterologous poll II promoter for the
promoter elements of the FIV LTR enabled high-level FIV protein
production, in trans, in human cells. In addition, selectively
replacing the FIV 5-prime U3 element by precisely fusing a
heterologous promoter to the R repeat resulted in high production
in human cells of wild type FIV that was replication-competent only
in feline cells. A three plasmid, replication-defective,
env-deleted, fully heterologously-promoted FIV vector system was
constructed and found to efficiently transduce dividing and
non-dividing human cells with vesicular stomatitis virus
glycoprotein G (VSV-G)-pseudotyped FIV particles. There was no
transduction advantage to feline cells; relative transduction
efficiencies in dividing cells of diverse human and feline lineages
were the same as for a Moloney murine leukemia virus (M-MuLV)-based
vector. In distinct contrast to the common M-MuLV vector, FIV
vectors efficiently transduced non-dividing human cells, including
terminally differentiated human macrophages and neurons (hNT
neurons). Extensive syncytia form when FIV expression is enabled in
human cells; this activity requires expression of CXCR4/fusin35,
the co-receptor for syncytium-inducing HIV isolates. Expression of
human CXCR4 in feline CRFK cells changes viral phenotype to highly
syncytium-inducing and mediates FIV-enveloped vector entry. The
studies show that FIV can utilize the human homologue of CXCR4 for
syncytiagenesis in human, rodent, and feline cells and, consistent
with a co-receptor role, for viral entry in feline cells.
[0052] Feline immunodeficiency virus that is replication-competent
for feline cells but not human cells is produced at high levels in
human and feline cells by a precise fusion of a heterologous
promoter such as the human cytomegalovirus immediate early promoter
(CMV promoter) and the FIV genome immediately upstream of the FIV R
repeat as described. In one embodiment, the fusion is joined
precisely over the TATA box (the TATA box of the CMV promoter is
used; FIV sequences begin at the Sac I site just downstream of the
TATA box). Details of this fusion, which preserves TATA Box to mRNA
transcriptional start site spatial arrangements, are diagrammed in
the examples.
[0053] FIV-derived retroviral vectors are optionally expressed at
high levels from the same heterologous promoter-R repeat fusion in
which the five-prime U3 element of the FIV vector has been
precisely replaced by a heterologous promoter. These FIV vectors
can be delivered at high titer in replication-defective fashion to
non-feline mammalian cells, including both dividing and
growth-arrested human cells, via heterologous envelopes, including
but not limited to pseudotyping by the vesicular stomatitis virus
envelope glycoprotein G (VSV-G); this is the first demonstration of
delivery of genes to human cells using a non-primate
lentivirus.
[0054] Co-production of FIV proteins and FIV vectors resulted in
high titer, replication-defective lentiviral vectors that can
deliver genes to human cells as well as feline cells. FIV vector
titers exceeding 107 per ml on human cells were achieved and higher
titers are achieved with refinements of production and
concentration using available methods. Therefore, the invention has
direct applicability to human gene therapy. Other promoters active
in human cells will also be useful in some embodiments.
[0055] The invention embodies several safety advantages. One
important safety advantage of this invention is production of the
replication-defective vector entirely in human cells. The native
FIV promoter (LTR) is inactive or minimally active in human cells.
In fact, the hCMVIE promoter as implemented here permits higher
expression than the FIV LTR in feline as well as human cells. While
the native FIV LTR is used in feline producer cells to drive
expression (analogously to the use of the Moloney murine leukemia
(MoMLV) virus promoter in MoMLV-based systems), such use presents
distinct safety problems that are preferably avoided. Feline cells
can contain a replication-competent type C endogenous retrovirus
(RD114) that replicates in human cells. Feline cells, which have
received less scientific study that human cells, may also contain
other unknown infectious agents that are potentially pathogenic.
RD114 was originally isolated from human rhabdomyosarcoma cells
that had been passaged in fetal kittens; replication-competent
RD114 has also been isolated from cultured feline cell lines by
co-cultivation with human and other non-feline cells. In addition,
RD144 is strikingly related at the nucleotide sequence level to a
primate retrovirus (baboon endogenous virus or BaEV); this fact, in
addition to demonstrated replication competence in human cell
lines, suggests clear potential for cross-species transmission to
humans. The danger of transmission of endogenous retroviruses such
as RD114 or other viruses to humans through xenotransplantation or
exposure to biological materials derived from animal cells or
tissues has recently generated great concern. Pig cells, for
example, have now been shown to harbor an endogenous retrovirus
that can replicate in human cells similarly to RD114; this finding
is now considered by experts in the field to raise serious doubts
about the safety of animal organ xenotransplantation into humans or
use for therapeutic purposes of animal-derived biologicals that are
not sterilizable. Therefore, exposure of humans to vectors derived
from feline or ungulate cell lines, which are far less well
characterized than human or rodent cell lines, would be
problematic. The chimeric design of the vectors herein pernits
production entirely in well-defined human producer cell lines such
as 293T cells. The safety advantage is avoidance of exposure at any
stage of production to feline cells or tissues. Accordingly, for
applications involving introduction of cells or vectors to
patients, it is preferable to use vectors packaged in human
packaging cells.
[0056] In all its embodiments, the system also represents a
significant safety advance over HIV-based lentiviral vectors
because, as noted above, FIV is neither transmissible to humans nor
pathogenic in humans. This invention therefore makes use of the
well-documented fact that no evidence for pathogenicity of FIV in
humans exists. The practical applicability of this invention also
benefits from the fact that lack of tropism or pathogenicity in
humans is better established for FIV than for any other non-primate
lentivirus (these include Visna/Maedi, CAEV, EIAV and BIV) because
many thousands of humans are exposed yearly by the same means by
which FIV is predominantly transmitted among both feral and
domestic cats in nature, i.e., cat bites.
[0057] All lentiviruses are transmitted exclusively by exchange of
body fluids. HIV is predominantly sexually transmitted; there is
little evidence that FIV is sexually transmitted in nature;
instead, cat bites--which humans are also commonly exposed to--are
the main mechanism. Bite wounds are common among cats because these
animals are territorial and males in particular engage in frequent
fights over territory and dominance; FIV infection is accordingly
more common among male cats than among female cats.
[0058] In summary, there is no evidence for FIV infection of
non-felids: this restricted tropism is characteristic of all known
lentiviruses. Biting is the major natural means of spread of FIV
between cats and despite frequent exposure of humans to FIV by cat
bites, this plausibly efficient means of inoculation does not
result in human seroconversion or any other detectable evidence of
human infection. Among the non-primate lentiviruses, therefore,
epidemiological evidence against human pathogenicity is compelling
for FIV.
[0059] Since FIV is routinely worked with using Biosafety Level-2
(BL-2) practices, vectors can be routinely worked with in BL-2
facilities, risks posed to personnel involved in their development
and production are lessened compared to HIV vectors, and ease and
convenience of production is enhanced.
[0060] In addition to the safety advantages inherent in using a
non-primate lentivirus for cellular gene transfer and gene therapy,
a preferred system described herein from a molecular clone of FIV
(34TF10) is defective in an important FIV gene (ORF2) and is
therefore an attenuated virus. 34TF10 replicates in adherent cat
cell lines but not in lymphocytes; replication-competence in feline
lymphocytes has been mapped to ORF2. In addition, the ORF2-minus
virus is neural tropic. If needed for delivery to certain cell
types, ORF2 can be repaired. In addition, in other embodiments,
other strains or molecular clones of FIV are used in a similar
fashion since the present invention makes apparent their
adaptability to transduction of human cells.
[0061] Although a chimeric construction fusing the heterologous
promoter to FIV sequences was used in one example herein, because
the fusion is located at the start of transcription, only FIV
sequences are transcribed (i.e., only FIV promoter sequences appear
in the mRNA generated by the system; the heterologous promoter
sequences are not transcribed). The possibility of a
replication-competent chimeric virus arising by RNA-level
recombination is therefore reduced.
[0062] In addition, since this lentivirus that is phylogentically
distant from HIV is now shown in the present invention to be
adaptable to transduction of human cells, it is now clear that
other non-primate lentiviruses, particularly the ungulate
lentiviruses, can be similarly utilized.
[0063] By engineering expression from the packaging plasmid, the
vector and the envelope expression plasmid occur from the same
human promoter, synchronized expression (i.e., temporal
coordination) of protein expression is facilitated.
[0064] Making Packaging Plasmids and Packageable Nucleic Acids
[0065] The present invention provides a variety of packaging
plasmids and packageable nucleic acids as described supra.
Packaging plasmids include non-primate lentiviral derived nucleic
acids, particularly those derived from FIV and the ungulate
lentiviruses. Packageable nucleic acid vectors encode RNAs which
comprise a lentiviral packaging site (e.g., FIV or ungulate
derived), and optionally comprise other non-primate lentiviral
nucleic acids, or heterologous nucleic acids.
[0066] The packagable vectors and packaging plasmids of the
invention are derived from lentiviral clones. Many such clones are
known to persons of skill, and publicly available. Well-established
repositories of sequence information include GenBank, EMBL, DDBJ
and the NCBI. Well characterized HIV clones include:
HIV-1.sub.NL43, HIV-1.sub.SF2, HIV-1.sub.BRU, HIV-1.sub.MN.
[0067] Furthermore, viral clones can be isolated from wild-type
viruses using known techniques. Typically, a lambda-phage clone,
containing a full-length lentiviral provirus, is obtained from the
genomic DNA of a cell line infected with a viral strain isolated
from the peripheral blood mononuclear cells of a seropositive
animal. The virus is replication competent in vitro, producing
infectious progeny virions after direct transfection into target
cells from the organism (e.g., CD4.sup.+ cells). In general, a
complete virulent genome can be used to make a packaging plasmid. A
"full-length FIV genome" in relation to an FIV packaging vector
consists of a nucleic acid (RNA or DNA) encoded by an FIV virus or
viral clone (e.g., a DNA phage) which includes the 5' and 3' LTR
regions and the genes between the LTR regions which are present in
a typical corresponding virus. For FIV, these genes include:
gag-pol, env and orf-2, and in addition there are several
uncharacterized reading frames, particularly in env.
[0068] A packaging plasmid is made by deleting the packaging site
from a full-length genome, rendering the clone capable of producing
viral proteins, but incapable of self packaging viral RNAs. The RNA
secondary structure of the FIV packaging site likely includes the
region between MSD and gag-pol; in addition, other regions may be
important for proper packaging. The precise nature of the packaging
site can be determined by performing deletion analysis.
[0069] In packaging plasmids, preferably, the entire psi site
(packaging site) is deleted, but any mutation or deletion which
deletes or mutates enough of the site to inhibit packaging is
sufficient. This typically results in a substantial deletion in the
region between the major splice donor site ("MSD") and the
beginning of the gag gene, and may include other regions as well.
Deletion of promoter elements which direct viral expression of the
encoded viral proteins is also desirable, as is incorporation of
heterologous promoters. In particular, human promoters (i.e.,
promoters active in human cells which are typically, but not
necessarily, of human origin, or derived from a human pathogen such
as a virus which is active in a human).
[0070] The resulting packaging plasmids of the invention are used
to make viral particles, by transducing the deletion clone into a
packaging cell (typically a human cell such as a 293 or other
well-characterized cell which does not comprise any unwanted
components) and expressing the plasmid. Because the plasmids lack a
lentiviral packaging site, they are not packaged into viral
particles.
[0071] To increase the safety of the transduced packaging cells, it
is preferable to cut (e.g., by subcloning) the packaging plasmid
(or homologous clones) into multiple packaging plasmids with
complementary functions. This decreases the chances that a
recombination event will result in an infectious particle.
[0072] Packageable nucleic acids encode an RNA which is competent
to be packaged by an FIV particle. Such nucleic acids can be
constructed by recombinantly combining an FIV packaging site with a
nucleic acid of choice. The packaging site (psi site) is located
adjacent to the 5' LTR, primarily between the MSD site and the gag
initiator codon (AUG) in the leader sequence of the gag gene. Thus,
the minimal packaging site includes a majority of nucleic acids
between the MSD and the gag initiator codon from the relevant
lentivirus. Preferably, a complete packaging site includes
sequences from the 5' LTR and the 5' region of gag gene for maximal
packaging efficiency. These packaging sequences typically include a
portion of the gag gene, e.g., extending about 100 bases into the
coding region of gag or further, and about 100 bases into the FIV
5' LTR or further. In addition, sequences from the env gene are
optionally included in the packaging site, particularly the
RRE.
[0073] Given the strategy for making the packaging plasmids and
target packageable vector nucleic acids of the present invention,
one of skill can construct a variety of clones containing
functionally equivalent nucleic acids. Cloning methodologies to
accomplish these ends, and sequencing methods to verify the
sequence of nucleic acids are well known in the art. Examples of
appropriate cloning and sequencing techniques, and instructions
sufficient to direct persons of skill through many cloning
exercises are found in Berger and Kimmel, Guide to Molecular
Cloning Techniques, Methods in Enzymology volume 152 Academic
Press, Inc., San Diego, Calif. (Berger); Sambrook et al. (1989)
Molecular Cloning--A Laboratory Manual (2nd ed.) Vol. 1-3, Cold
Spring Harbor Laboratory, Cold Spring Harbor Press, NY, (Sambrook);
and Current Protocols in Molecular Biology, F. M. Ausubel et al.,
eds., Current Protocols, a joint venture between Greene Publishing
Associates, Inc. and John Wiley & Sons, Inc., (1994 Supplement)
(Ausubel). Product information from manufacturers of biological
reagents and experimental equipment also provide information useful
in known biological methods. Such manufacturers include the SIGMA
chemical company (Saint Louis, Mo.), R&D systems (Minneapolis,
Minn.), Pharmacia LKB Biotechnology (Piscataway, N.J.), CLONTECH
Laboratories, Inc. (Palo Alto, Calif.), Chem Genes Corp., Aldrich
Chemical Company (Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL
Life Technologies, Inc. (Gaithersberg, Md.), Fluka
Chemica-Biochemika Analytika (Fluka Chemie A G, Buchs,
Switzerland), Invitrogen, San Diego, Calif., and Applied Biosystems
(Foster City, Calif.), as well as many other commercial sources
known to one of skill.
[0074] The nucleic acid compositions of this invention, whether
RNA, cDNA, genomic DNA, or a hybrid of the various combinations,
are isolated from biological sources or synthesized in vitro. The
nucleic acids of the invention are present in transformed or
transfected whole cells, in transformed or transfected cell
lysates, or in a partially purified or substantially pure form.
[0075] In vitro amplification techniques suitable for amplifying
sequences for use as molecular probes or generating nucleic acid
fragments for subsequent subcloning are known. Examples of
techniques sufficient to direct persons of skill through such in
vitro amplification methods, including the polymerase chain
reaction (PCR) the ligase chain reaction (LCR), Q.beta.-replicase
amplification and other RNA polymerase mediated techniques (e.g.,
NASBA) are found in Berger, Sambrook, and Ausubel, as well as
Mullis et al., (1987) U.S. Pat. No. 4,683,202; PCR Protocols A
Guide to Methods and Applications (Innis et al. eds) Academic Press
Inc. San Diego, Calif. (1990) (Innis); Arnheim & Levinson (Oct.
1, 1990) C&EN 36-47; The Journal Of NIH Research (1991) 3,
81-94; (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86, 1173;
Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87, 1874; Lomell
et al. (1989) J. Clin. Chem 35, 1826; Landegren et al., (1988)
Science 241, 1077-1080; Van Brunt (1990) Biotechnology 8, 291-294;
Wu and Wallace, (1989) Gene 4, 560; Barringer et al. (1990) Gene
89, 117, and Sooknanan and Malek (1995) Biotechnology 13: 563564.
Improved methods of cloning in vitro amplified nucleic acids are
described in Wallace et al., U.S. Pat. No. 5,426,039.
[0076] Oligonucleotides for use as probes, e.g., in in vitro
amplification methods, for use as gene probes, or as inhibitor
components (e.g., ribozymes) are typically synthesized chemically
according to the solid phase phosphoramidite triester method
described by Beaucage and Caruthers (1981), Tetrahedron Letts.,
22(20): 1859-1862, e.g., using an automated synthesizer, as
described in Needham-VanDevanter et al. (1984) Nucleic Acids Res.,
12:6159-6168. Oligonucleotides can also be custom made and ordered
from a variety of commercial sources known to persons of skill.
Purification of oligonucleotides, where necessary, is typically
performed by either native acrylamide gel electrophoresis or by
anion-exchange HPLC as described in Pearson and Regnier (1983) J.
Chrom. 255:137-149. The sequence of the synthetic oligonucleotides
can be verified using the chemical degradation method of Maxam and
Gilbert (1980) in Grossman and Moldave (eds.) Academic Press, New
York, Methods in Enzymology 65:499-560.
[0077] Making Conservative Substitutions
[0078] One of skill will appreciate that many conservative
variations of the nucleic acid constructs disclosed yield a
functionally identical construct. For example, due to the
degeneracy of the genetic code, "silent substitutions" (i.e.,
substitutions of a nucleic acid sequence which do not result in an
alteration in an encoded polypeptide) are an implied feature of
every nucleic acid sequence which encodes an amino acid. Similarly,
"conservative amino acid substitutions," in one or a few amino
acids in an amino acid sequence of a packaging or packageable
construct are substituted with different amino acids with highly
similar properties (see, the definitions section, supra), are also
readily identified as being highly similar to a disclosed
construct. Such conservatively substituted variations of each
explicitly disclosed sequence are a feature of the present
invention.
[0079] One of skill will recognize many ways of generating
alterations in a given nucleic acid construct. Such well-known
methods include site-directed mutagenesis, PCR amplification using
degenerate oligonucleotides, exposure of cells containing the
nucleic acid to mutagenic agents or radiation, chemical synthesis
of a desired oligonucleotide (e.g., in conjunction with ligation
and/or cloning to generate large nucleic acids) and other
well-known techniques. See, Giliman and Smith (1979) Gene 8:81-97,
Roberts et al. (1987) Nature 328:731-734 and Sambrook, Innis,
Ausbel, Berger, Needham VanDevanter and Mullis (all supra).
[0080] One of skill can select a desired nucleic acid of the
invention based upon the sequences provided and upon knowledge in
the art regarding retroviruses generally. The specific effects of
many mutations in retroviral genomes are known. Moreover, general
knowledge regarding the nature of proteins and nucleic acids allows
one of skill to select appropriate sequences with activity similar
or equivalent to the nucleic acids and polypeptides disclosed in
the sequence listings herein. The definitions section herein
describes exemplar conservative amino acid substitutions.
[0081] Finally, most modifications to nucleic acids are evaluated
by routine screening techniques in suitable assays for the desired
characteristic. For instance, changes in the immunological
character of encoded polypeptides can be detected by an appropriate
immunological assay. Modifications of other properties such as
nucleic acid hybridization to a complementary nucleic acid, redox
or thermal stability of encoded proteins, hydrophobicity,
susceptibility to proteolysis, or the tendency to aggregate are all
assayed according to standard techniques.
[0082] Making Stable Packaging Cell Lines
[0083] Stable packaging cell lines are made by stably or
transiently transducing a mammalian cell with a packaging plasmid,
most preferably by transducing a human cell. The transduction of
mammalian (including human) cells is known in the art. Host cells
are competent or rendered competent for transformation by various
known means. There are several well-known methods of introducing
DNA into animal cells. These include: calcium phosphate
precipitation, fusion of the recipient cells with bacterial
protoplasts containing the DNA, treatment of the recipient cells
with liposomes containing the DNA, DEAE dextran, receptor-mediated
endocytosis, electroporation and micro-injection of the DNA
directly into the cells.
[0084] The culture of cells used in conjunction with the present
invention, including cell lines and cultured cells from tissue or
blood samples is well known in the art. Freshney (Culture of Animal
Cells, a Manual of Basic Technique, third edition Wiley-Liss, New
York (1994)) and the references cited therein provides a general
guide to the culture of cells. Transformed cells are cultured by
means well known in the art. See, also Kuchler et al. (1977)
Biochemical Methods in Cell Culture and Virology, Kuchler, R. J.,
Dowden, Hutchinson and Ross, Inc. Mammalian cell systems often will
be in the form of monolayers of cells, although mammalian cell
suspensions are also used. Illustrative examples of mammalian cell
lines include VERO and HeLa cells, 293 embryonic kidney cells,
Chinese hamster ovary (CHO) cell lines, W138, BHK, Cos-7 or MDCK
cell lines (see, e.g., Freshney, supra). Human cells are most
preferred.
[0085] Supernatants from cell cultures of the packaging cells of
the invention are obtained using standard techniques such as those
taught in Freshney, supra. See also, Corbeau et al. (1996) Proc.
Natl. Acad. Sci. USA 93:14070-14075 and the references therein.
Components from the cell supernatants can be further purified using
standard techniques. For example, FIV particles in the supernatant
can be purified from the supernatant by methods typically used for
viral purification such as centrifugation, chromatography, affinity
purification procedures, and the like.
[0086] Transforming mammalian cells with nucleic acids can involve,
for example, incubating competent cells with a plasmid containing
nucleic acids which code for an FIV particle. The plasmid which is
used to transform the host cell preferably contains nucleic acid
sequences to initiate transcription and sequences to control the
translation of the encoded sequences. These sequences are referred
to generally as expression control sequences. Illustrative
mammalian expression control sequences are obtained from the SV-40
promoter (Science (1983) 222:524-527), the CMV I.E. Promoter (Proc.
Natl. Acad. Sci. (1984) 81:659-663), the CMV promoter, or the
metallothionein promoter (Nature (1982) 296:39-42). A cloning
vector containing expression control sequences is cleaved using
restriction enzymes and adjusted in size as necessary or desirable
and ligated with DNA coding for the HIV sequences of interest by
means well known in the art. A huge variety of specific pol II and
pol III promoters active in human cells are known in the art and
one of skill is able to select and use such promoters based upon
the desired expression level, pattern, transformed cell, or the
like.
[0087] Polyadenlyation or transcription terminator sequences from
known mammalian genes are typically incorporated into the vectors
of the invention. An example of a terminator sequence is the
polyadenlyation sequence from the bovine growth hormone gene.
Sequences for accurate splicing of the transcript may also be
included. An example of a splicing sequence is the VP1 intron from
SV40 (Sprague et al. (1983) J. Virol. 45: 773-781). Additionally,
gene sequences to control replication in a particular host cell are
incorporated into the vector such as those found in bovine
papilloma virus type-vectors. See, Saveria-Campo (1985), "Bovine
Papilloma virus DNA a Eukaryotic Cloning Vector" in DNA Cloning
Vol. II a Practical Approach Glover (ed) IRL Press, Arlington, Va.
pp. 213-238.
[0088] Co-plasmids can be used in selection methods. In these
methods, a plasmid containing a selectable marker, such as an
antibiotic resistance gene, is used to co-transfect a cell in
conjunction with a plasmid encoding HIV packaging nucleic acids.
The cells are selected for antibiotic resistance, and the presence
of the plasmid of interest is confirmed by Southern analysis,
northern analysis, or PCR. Co-plasmids encoding proteins to be
expressed on the surface of an HIV particle (e.g., proteins which
expand the host range of the capsid such as the VSV envelope, a
cell receptor ligand, or an antibody to a cell receptor) are
optionally transduced into the packaging cell. In addition to VSV,
the envelope proteins of other lipid enveloped viruses are
optionally incorporated into a particle of the invention, thereby
expanding the transduction range of the particle.
[0089] Viral vectors containing nucleic acids which encode selected
sequences are also used to transform cells within the host range of
the vector. See, e.g., Methods in Enzymology, vol. 185, Academic
Press, Inc., San Diego, Calif. (D. V. Goeddel, ed.) (1990) or M.
Krieger, Gene Transfer and Expression--A Laboratory Manual,
Stockton Press, New York, N.Y., (1990) and the references cited
therein.
[0090] Once stable transformed cell lines are made which express
FIV particles, the transformed cell lines are transfected with
vectors which encode nucleic acids to be incorporated into the FIV
vectors. Typically, these vectors are plasmids or are coded in
viral vectors. The packaged nucleic acids include an FIV packaging
site subsequence in conjunction with a sequence of interest, such
as a viral inhibitor or other gene therapeutic (it will be
appreciated that any condition which is meditated by non-dividing
cells or their progeny are treatable using the vectors of the
invention, including HIV infections, HTLV infections, lymphomas,
leukemias, neural tumors and the like).
[0091] Human cells are preferred packaging cell lines, and can be
made competent for transformation by the techniques described
above. For example, to generate a stable FIV packaging cell line,
FIV cells were transfected by the Calcium-Phosphate method as
described in the examples herein. A subconfluent culture, e.g., in
a 6-well plate (Costar, Cambridge, Mass.) is transfected with
linearized and calcium-phosphate precipitated plasmid (10 .mu.g),
e.g., in Dulbecco's modified Eagle's medium supplemented with 10%
FCS, antibiotics and glutamine (DMEM-10%FCS), optionally with a
co-plasmid marker. After 18 hours, wells are washed with Dulbecco's
phosphate-buffered saline (PBS) pH 7.8, incubated for 2 min. at
20.degree. C. with 15% glycerol solution in HEPES-buffered saline
(50 mM HEPES pH 7.1, 280 mM NaCl, 1.5 mM Na2HP04), washed twice
with PBS and cultured in DMEM-10%FCS. The cells are subjected to
selection as appropriate for a transduction marker gene.
[0092] Assaying for Packaging Plasmids, Packageable Nucleic Acids
and FIV Particles in Packaging Cell Lines, Target Cells and Cell
Lysates
[0093] A wide variety of formats and labels are available and
appropriate for detection of packaging plasmids, packageable
nucleic acids and lentiviral particles in packaging cells, target
cells, patients and cell lysates. Antibodies to lentiviral
components, and the polypeptides and nucleic acids of the invention
(vectors, packaging plasmids, encoded nucleic acids and
polypeptides, etc.) are detected and quantified by any of a number
of means well known to those of skill in the art. These include
analytic biochemical methods such as spectrophotometry,
radiography, electrophoresis, capillary electrophoresis, high
performance liquid chromatography (HPLC), thin layer chromatography
(TLC), hyperdiffusion chromatography, and the like, and various
immunological methods such as fluid or gel precipitin reactions,
immunodiffusion (single or double), immunoelectrophoresis,
radioimmunoassays (RIAs), enzyme-linked immunosorbent assays
(ELISAs), immunofluorescent assays, and the like. Several available
ELISA assays for the detection of lentiviral components (e.g., FIV)
are available, allowing one of skill to detect non-primate
lentiviral particles, or non-primate lentiviral virus in a
biological sample.
[0094] The detection of nucleic acids proceeds by well known
methods such as Southern analysis, northern analysis, gel
electrophoresis, PCR, radiolabeling and scintillation counting, and
affinity chromatography. Many assay formats are appropriate,
including those reviewed in Tijssen (1993) Laboratory Techniques in
biochemistry and molecular biology--hybridization with nucleic acid
probes parts I and II, Elsevier, New York and Choo (ed) (1994)
Methods In Molecular Biology Volume 33--In Situ Hybridization
Protocols Humana Press Inc., New Jersey (see also, other books in
the Methods in Molecular Biology series); see especially, Chapter
21 of Choo (id) "Detection of Virus Nucleic Acids by Radioactive
and Nonisotopic in Situ Hybridization". A variety of automated
solid phase detection techniques are also appropriate. For
instance, very large scale immobilized polymer arrays (VLSIPS.TM.)
are used for the detection of nucleic acids. See, Tijssen (supra),
Fodor et al. (1991) Science, 251: 767-777 and Sheldon et al. (1993)
Clinical Chemistry 39(4): 718-719. Finally, PCR is also routinely
used to detect nucleic acids in biological samples (see, Innis,
supra for a general description of PCR techniques).
[0095] In one preferred embodiment, antibodies are used to detect
proteins expressed by the packaged vector, the packaging nucleic
acid or to monitor circulating HIV, HTLV (or other relevant
pathogen), levels in human blood, e.g., to monitor the in vivo
effect of a gene therapeutic agent coded by the FIV packaged
nucleic acids. In other embodiments, antibodies are co-expressed in
the packaging cells to be incorporated into viral particles.
Methods of producing polyclonal and monoclonal antibodies are known
to those of skill in the art, and many antibodies are available.
See, e.g., Coligan (1991) Current Protocols in Immunology
Wiley/Greene, NY; and Harlow and Lane (1989) Antibodies: A
Laboratory Manual Cold Spring Harbor Press, NY; Stites et al.
(eds.) Basic and Clinical Immunology (4th ed.) Lange Medical
Publications, Los Altos, Calif., and references cited therein;
Goding (1986) Monoclonal Antibodies: Principles and Practice (2d
ed.) Academic Press, New York, N.Y.; and Kohler and Milstein (1975)
Nature 256: 495-497. Other suitable techniques for antibody
preparation include selection of libraries of recombinant
antibodies in phage or similar vectors. See, Huse et al. (1989)
Science 246: 1275-1281; and Ward, et al. (1989) Nature 341:
544-546. Specific monoclonal and polyclonal antibodies and antisera
will usually bind with a KD of at least about 0.1 .mu.M, preferably
at least about 0.01 .mu.M or better, and most typically and
preferably, 0.001 .mu.M or better.
[0096] Use of the Nucleic acids of the Invention as Molecular
Probes
[0097] In addition to their utility in making packaging cell lines,
the non-infective packaging plasmids of the invention can be used
to detect wild-type virus in biological samples using Southern or
northern blot assays. In brief, a nucleic acid encoded by the
packaging plasmid is labeled, typically using a radio or
bioluminescent label, and used to probe a northern or Southern blot
of a sample suspected of containing a virus (FIV, an ungulate
retrovirus, etc.). The use of the packaging plasmid as a probe is
safer than the use of an infective virus as a probe. The packaging
plasmid is also more likely to detect a wild type virus than a
smaller probe, because, unlike a small probe, the packaging plasmid
probe optionally has virtually the entire genome in common with a
wild-type virus (except for the packaging site), making it
improbable that the wild type virus could escape detection by
mutation of the probe binding site.
[0098] Furthermore, the packaging plasmid can be used as positive
controls in essentially all known detection methods for the
detection of the corresponding retrovirus (e.g., FIV). In this
embodiment, a packaging plasmid nucleic acid or encoded polypeptide
is used as a positive control to establish that an FIV detection
assay is functioning properly. For instance, oligonucleotides are
used as primers in PCR reactions to detect FIV nucleic acids in
biological samples such as feline blood in veterinary settings. The
packaging plasmid, which comprises nucleic acid subsequences
corresponding to the region to be amplified is used as an
amplification templates in a separate reaction from a test sample
such as human blood to determine that the PCR reagents and
hybridization conditions are appropriate. Similarly, the
polypeptides encoded by the packaging plasmid can be used to check
ELISA reagents in assays for the detection of FIV expression
products in biological samples.
[0099] Packageable nucleic acids can also be used in the same
fashion as molecular probes, e.g., when they encode HIV components
such as HIV packaging sites, HIV LTRs, transdominant Tat or Rev
proteins, or the like, for detection of HIV, or as HIV detection
reagents.
[0100] Cellular Transformation and Gene Therapy
[0101] The present invention provides packageable nucleic acids for
the transformation of cells in vitro and in vivo. These packageable
nucleic acids are packaged in non-primate lentiviral particles such
as FIV particles, in the lentiviral packaging cell lines described
herein. Preferably, when the target is in the host range of the VSV
virus (e.g., a hematopoietic stem cell) the particle also includes
VSV glycoproteins. The nucleic acids are transfected into cells
through the interaction of the FIV particle surrounding the nucleic
acid and an FIV or VSV cellular receptor.
[0102] In one particularly preferred class of embodiments, the
packageable nucleic acids of the invention are used in cell
transformation procedures for gene therapy. Gene therapy provides
methods for combating chronic infectious diseases such as HIV, as
well as non-infectious diseases such as cancer and birth defects
such as enzyme deficiencies. Yu et al. (1994) Gene Therapy 1:13-26
and the references therein provides a general guide to gene therapy
strategies for HIV infection. See also, Sodoski et al.
PCT/US91/04335. One general limitation of common gene therapy
vectors such as murine retroviruses is that they only infect
actively dividing cells, and they are generally non-specific. The
present invention provides several features that allow one of skill
to generate powerful retroviral gene therapy vectors which
specifically target stem cells in vivo, and which transform many
cell types in vitro. CD4.sup.+ cells, neuronal cells and other
non-dividing cells (often CXCR4 positive) are transduced by nucleic
acids packaged in FIV particles. In addition, the vectors are
optionally pseudotyped for transformation of stem cells.
[0103] Pseudotyping the Packageable Vector
[0104] Hematopoietic stem cells are particularly preferred targets
for cell transformation in general, and for gene therapy
(particularly anti-HIV gene therapy) in particular. Packageable
vectors are made competent to transform CD34.sup.+ cells by
pseudotyping the vector. This is done by transducing the packaging
cell line used to package the vector with a nucleic acid which
encodes an Env protein which supplants or complements the
retroviral env function. The envelope function can be supplied in
trans by any number of heterologous viral envelope proteins. These
include, but are not limited to, VSV-G, the arnphotropic envelope
of Moloney murine leukemia virus (MoMuLV), and gibbon ape leukemia
virus (GALV) envelope. The vesicular stomatitis virus (VSV)
envelope glycoprotein, which expressed on the surface of the vector
is a preferred pseudotyping component. VSV infects both dividing
and non-dividing CD34.sup.+ cells, and pseudotype vectors
expressing VSV envelope proteins are competent to transduce these
cells.
[0105] Similarly, viral or cellular proteins in general can be
co-expressed to increase the host range of an FIV-based vector.
Typically, a nucleic acid encoding a selected protein is
coexpressed in an FIV packaging cell of the invention. Protein
encoded by the nucleic acid is incorporated into the particle which
packages an FIV-packageable nucleic acid, which buds off from the
packaging cell membrane. If the protein is recognized by a cellular
receptor on a target cell, the particle is transduced into the cell
by receptor mediated endocytosis. Preferred proteins include viral
envelope or coat proteins, cell receptor ligands, antibodies or
antibody fragments which bind cell receptors on target cells, and
the like.
[0106] Preferred Promoters
[0107] One class of embodiments utilizes an HIV LTR sequence as a
promoter for the FIV packageable vector. These LTR sequences are
trans-activated upon infection of a cell containing the LTR
promoter by the infecting HIV virus. HIV LTR promoters, in addition
to binding tat and rev are responsive to cellular cytokines (such
as IL-2 and SP-1) which act to permit transcription of the HIV
genome upon infection. Thus, in one embodiment, a therapeutic
nucleic acid of choice is placed under the control of an LTR
promoter, rendering the cells ordinarily most vulnerable to HIV
infection resistant to infection. Furthermore, in one embodiment,
an HIV packaging site (in addition to the FIV or other non-primate
lentiviral packaging site) is included in the FIV packageable
vector. This permits the anti-HIV therapeutic agent encoded in the
FIV based vector to be packaged and disseminated by infecting HIV
viruses, causing a secondary protective effect to be propagated in
combination with HIV infection, thereby slowing the infection. The
HIV packaging site is well described, see, Poznansky et al. (1991)
Journal or Virology 65(1): 532-536; Aldovini and Young (1990)
Journal of Virology 64(5): 1920-1926, and Clever et al. (1995)
Journal of Virology 69(4): 2101-2109. For a description of vectors
which are packaged by HIV to provide a secondary protective effect,
see, Wong-Stall et al. U.S. Pat. No. 5,650,309.
[0108] Constitutive promoters for directing expression of
therapeutic nucleic acids are also preferred, such as pol III
promoters. PCT application PCT/US94/05700 (WO 94/26877) and
Chatterjee et al. (Science (1992), 258: 1485-1488, hereinafter
Chatterjee et al. 2) describe anti-sense inhibition of HIV-1
infectivity in target cells using viral vectors with a constitutive
pol III expression cassette expressing anti-TAR RNA. Chatterjee et
al. (PCT application PCT/US91/03440 (1991), hereinafter Chatterjee
et al. 2) describe viral vectors, including AAV-based vectors which
express antisense TAR sequences. Chatterjee and Wong (Methods, A
companion to Methods in Enzymology (1993), 5: 51-59) further
describe viral vectors for the delivery of antisense RNA. PCT
publication WO 94/26877 (PCT/US94/05700) describes a variety of
anti-HIV therapy genes, and gene therapy strategies generally,
including the use of suicide genes, transdominant genes, ribozymes,
anti-sense genes, and decoy genes in gene therapy vectors. Yu et
al. (1994) Gene Therapy 1: 13-26 and the references cited therein
provides a general guide to gene therapy strategies useful against
HIV infection.
[0109] Er Vivo Transformation of Cells
[0110] Ex vivo methods for inhibiting viral replication in a cell
in an organism (or otherwise introducing a therapeutic gene into
the cell) involve transducing the cell ex vivo with a therapeutic
nucleic acid of this invention, and introducing the cell into the
organism. Target cells include CD4.sup.+ cells such as CD4.sup.+ T
cells or macrophage isolated or cultured from a patient, stem
cells, or the like. See, e.g., Freshney et al., supra and the
references cited therein for a discussion of how to isolate and
culture cells from patients. Alternatively, the cells can be those
stored in a cell bank (e.g., a blood bank). In one class of
preferred embodiments, the packageable nucleic acid encodes an
anti-viral therapeutic agent (e.g., suicide gene, trans-dominant
gene, anti-HIV ribozyme, anti-sense gene, or decoy gene) which
inhibits the growth or replication of an HIV virus, under the
control of an activated or constitutive promoter. The cell
transformation vector inhibits viral replication in any of those
cells already infected with HIV virus, in addition to conferring a
protective effect to cells which are not infected by HIV. In
addition, in preferred embodiments, the vector is replicated and
packaged into HIV capsids using the HIV replication machinery,
thereby causing the anti-HIV therapeutic gene to propagate in
conjunction with the replication of an HIV virus. Thus, an organism
infected with HIV can be treated for the infection by transducing a
population of its cells with a vector of the invention and
introducing the transduced cells back into the organism as
described herein. Thus, the present invention provides a method of
protecting cells in vitro, ex vivo or in vivo, even when the cells
are already infected with the virus against which protection is
sought.
[0111] In one particularly preferred embodiment, stem cells (which
are typically not CD4.sup.+) are used in ex-vivo procedures for
cell transformation and gene therapy. The advantage to using stem
cells is that they can be differentiated into other cell types in
vitro, or can be introduced into a mammal (such as the donor of the
cells) where they will engraft in the bone marrow. Methods for
differentiating CD34.sup.+ cells in vitro into clinically important
immune cell types using cytokines such a GM-CSF, IFN-.gamma., and
TNF-.alpha. are known (see, Inaba et al. (1992) J. Exp. Med. 176,
1693-1702, and Szabolcs et al. (1995) 154: 5851-5861). Methods of
pseudotyping FIV vectors so that they can transform stem cells are
described above. An affinity column isolation procedure can be used
to isolate cells which bind to CD34, or to antibodies bound to
CD34. See, Ho et al. (1995) Stem Cells 13 (suppl. 3): 100-105. See
also, Brenner (1993) Journal of Hematotherapy 2: 7-17. In another
embodiment, hematopoietic stem cells are isolated from fetal cord
blood. Yu et al. (1995) PNAS 92: 699-703 describe a preferred
method of transducing CD34.sup.+ cells from human fetal cord blood
using retroviral vectors. Rather than using stem cells, T cells are
also transduced in preferred embodiments in ex vivo procedures.
Several techniques are known for isolating T cells. In one method,
Ficoll-Hypaque density gradient centrifugation is used to separate
PBMC from red blood cells and neutrophils according to established
procedures. Cells are washed with modified AIM-V (which consists or
AIM-V (GIBCO) with 2 mM glutamine, 10 .mu.g/ml gentamicin sulfate,
50 .mu.g/ml streptomycin) supplemented with 1% fetal bovine serum
(FBS). Enrichment for T cells is performed by negative or positive
selection with appropriate monoclonal antibodies coupled to columns
or magnetic beads according to standard techniques. An aliquot of
cells is analyzed for desired cell surface phenotype (e.g., CD4,
CD8, CD3, CD14, etc.).
[0112] In general, the expression of surface markers facilitates
identification and purification of T cells. Methods of
identification and isolation of T cells include FACS, column
chromatography, panning with magnetic beads, western blots,
radiography, electrophoresis, capillary electrophoresis, high
performance liquid chromatography (HPLC), thin layer chromatography
(TLC), hyperdiffusion chromatography, and the like, and various
immunological methods such as fluid or gel precipitin reactions,
immunodiffusion (single or double), immunoelectrophoresis,
radioimmunoassays (RIAs), enzyme-linked immunosorbent assays
(ELISAs), immunofluorescent assays, and the like. For a review of
immunological and immunoassay procedures in general, see Stites and
Terr (eds.) 1991 Basic and Clinical Immunology (7th ed.) and Paul
supra. For a discussion of how to make antibodies to selected
antigens see, e.g. Coligan (1991) Current Protocols in Immunology
Wiley/Greene, NY; and Harlow and Lane (1989) Antibodies: A
Laboratory Manual Cold Spring Harbor Press, NY; Stites et al.
(eds.) Basic and Clinical Immunology (4th ed.)
[0113] In addition to the ex vivo uses described above, the
packaging cell lines of the invention and the HIV packageable
nucleic acids of the invention are useful generally in cloning
methods. Packageable nucleic acids are packaged in an FIV particle
and used to transform an FIV-infectible cell (e.g., a feline
hematopoietic cell) in vitro or in vivo. This provides one of skill
with a technique and vectors for transforming cells with a nucleic
acid of choice, e.g., in drug discovery assays, or as a tool in the
study of gene regulation, or as a general cloning vector.
[0114] In Vivo Transformation
[0115] Non-primate lentiviral particles containing therapeutic
nucleic acids can be administered directly to the organism for
transduction of cells in vivo. Administration is by any of the
routes normally used for introducing a molecule into ultimate
contact with blood or tissue cells. Packageable nucleic acids
packaged in FIV or other non-primate lentiviral particles are used
to treat and prevent virally-mediated diseases such as AIDS in
animals and human patients. The packaged nucleic acids are
administered in any suitable manner, preferably with
pharmaceutically acceptable carriers. Suitable methods of
administering such packaged nucleic acids in the context of the
present invention to a patient are available, and, although more
than one route can be used to administer a particular composition,
a particular route can often provide a more immediate and more
effective reaction than another route.
[0116] Pharmaceutically acceptable carriers are determined in part
by the particular composition being administered, as well as by the
particular method used to administer the composition. Accordingly,
there is a wide variety of suitable formulations of pharmaceutical
compositions of the present invention.
[0117] The packaged nucleic acids, alone or in combination with
other suitable components, can also be made into aerosol
formulations (i.e., they can be "nebulized") to be administered via
inhalation. Aerosol formulations can be placed into pressurized
acceptable propellants, such as dichlorodifluoromethane, propane,
nitrogen, and the like.
[0118] Formulations suitable for parenteral administration, such
as, for example, by intraarticular (in the joints), intravenous,
intramuscular, intradermal, intraperitoneal, and subcutaneous
routes, include aqueous and non-aqueous, isotonic sterile injection
solutions, which can contain antioxidants, buffers, bacteriostats,
and solutes that render the formulation isotonic with the blood of
the intended recipient, and aqueous and non-aqueous sterile
suspensions that can include suspending agents, solubilizers,
thickening agents, stabilizers, and preservatives. Parenteral
administration and intravenous administration are the preferred
methods of administration. The formulations of packaged nucleic
acid can be presented in unit-dose or multi-dose sealed containers,
such as ampules and vials.
[0119] Cells transduced by the packaged nucleic acid as described
above in the context of ex vivo therapy can also be administered
intravenously or parenterally as described above.
[0120] The dose administered to a patient, in the context of the
present invention should be sufficient to effect a beneficial
therapeutic response in the patient over time, or to inhibit
infection by a pathogen. The dose will be determined by the
efficacy of the particular vector employed and the condition of O
the patient, as well as the body weight or surface area of the
patient to be treated. The size of the dose also will be determined
by the existence, nature, and extent of any adverse side-effects
that accompany the administration of a particular vector, or
transduced cell type in a particular patient.
[0121] In determining the effective amount of the vector to be
administered in the treatment or prophylaxis of virally-mediated
diseases such as AIDS, the physician evaluates circulating plasma
levels, vector toxicities, progression of the disease, and the
production of anti-vector antibodies. In general, the dose
equivalent of a naked nucleic acid from a vector is from about 1
.mu.g to 100 .mu.g for a typical 70 kilogram patient, and doses of
vectors which include a retroviral particle are calculated to yield
an equivalent amount of inhibitor nucleic acid. The vectors of this
invention can supplement treatment of virally-mediated conditions
by any known conventional therapy, including cytotoxic agents,
nucleotide analogues and biologic response modifiers.
[0122] For administration, inhibitors and transduced cells of the
present invention can be administered at a rate determined by the
LD-50 of the inhibitor, vector, or transduced cell type, and the
side-effects of the inhibitor, vector or cell type at various
concentrations, as applied to the mass and overall health of the
patient. Administration can be accomplished via single or divided
doses.
[0123] For introduction of transduced cells, prior to infusion,
blood samples are obtained and saved for analysis. Between
1.times.10.sup.8 and 1.times.10.sup.2 transduced cells are infused
intravenously over 60-200 minutes. Vital signs and oxygen
saturation by pulse oximetry are closely monitored. Blood samples
are obtained 5 minutes and 1 hour following infusion and saved for
subsequent analysis. Leukopheresis, transduction and reinfusion are
repeated every 2 to 3 months for a total of 4 to 6 treatments in a
one year period. After the first treatment, infusions can be
performed on a outpatient basis at the discretion of the clinician.
If the reinfusion is given as an outpatient, the participant is
monitored for at least 4, and preferably 8 hours following the
therapy.
[0124] Transduced cells are prepared for reinfusion according to
established methods. See, Abrahamsen et al. (1991) J. Clin.
Apheresis 6:48-53; Carter et al. (1988) J. Clin. Arpheresis
4:113-117; Aebersold et al. (1988), J. Immunol. Methods 112: 1-7;
Muul et al. (1987) J. Immunol. Methods 101:171-181 and Carter et
al. (1987) Transfusion 27:362-365. After a period of about 2-4
weeks in culture, the cells should number between 1.times.10.sup.8
and 1.times.10.sup.12. In this regard, the growth characteristics
of cells vary from patient to patient and from cell type to cell
type. About 72 hours prior to reinfusion of the transduced cells,
an aliquot is taken for analysis of phenotype, and percentage of
cells expressing the therapeutic agent.
[0125] If a patient undergoing infusion of a vector or transduced
cell develops fevers, chills, or muscle aches, he/she receives the
appropriate dose of aspirin, ibuprofen or acetaminophen. Patients
who experience reactions to the infusion such as fever, muscle
aches, and chills are premedicated 30 minutes prior to the future
infusions with either aspirin, acetaminophen, or diphenhydramine.
Meperidine is used for more severe chills and muscle aches that do
not quickly respond to antipyretics and antihistamines. Cell
infusion is slowed or discontinued depending upon the severity of
the reaction.
[0126] Viral Inhibitors
[0127] Specialized viral inhibitors are typically encoded by the
packaged nucleic acids of the invention, where the intended use is
viral (e.g., HIV) inhibition. Thus, techniques applicable to the
construction and maintenance of nucleic acids apply to the
inhibitors of the present invention. Anti-viral agents which are
optionally incorporated into the viral inhibitors of the invention
include anti-sense genes, ribozymes, decoy genes, and transdominant
nucleic acids.
[0128] An antisense nucleic acid is a nucleic acid that, upon
expression, hybridizes to a particular RNA molecule, to a
transcriptional promoter or to the sense strand of a gene. By
hybridizing, the antisense nucleic acid interferes with the
transcription of a complementary nucleic acid, the translation of
an mRNA, or the function of a catalytic RNA. Antisense molecules
useful in this invention include those that hybridize to viral gene
transcripts. Two target sequences for antisense molecules are the
first and second exons of the HIV genes tat and rev. Chatterjee and
Wong, supra, and Marcus-Sekura (Analytical Biochemistry (1988) 172,
289-285) describe the use of anti-sense genes which block or modify
gene expression.
[0129] A ribozyme is a catalytic RNA molecule that cleaves other
RNA molecules having particular nucleic acid sequences. General
methods for the construction of ribozymes, including hairpin
ribozymes, hammerhead ribozymes, RNAse P ribozymes (i.e., ribozymes
derived from the naturally occurring RNAse P ribozyme from
prokaryotes or eukaryotes) are known in the art. Castanotto et al
(1994) Advances in Pharmacology 25: 289-317 provides and overview
of ribozymes in general, including group I ribozymes, hammerhead
ribozymes, hairpin ribozymes, RNAse P, and axhead ribozymes.
Ribozymes useful in this invention include those that cleave viral
transcripts, particularly HIV gene transcripts. Ojwang et al.,
Proc. Nat'l. Acad. Sci., U.S.A., 89:10802-06 (1992); Wong-Staal et
al. (PCT/US94/05700); Ojwang et al. (1993) Proc Natl Acad Sci USA
90:6340-6344; Yamada et al. (1994) Human Gene Therapy 1:39-45,
Leavitt et al. (1995) Proc Natl Acad Sci USA 92:699-703; Leavitt et
al. (1994) Human Gene Therapy 5:1151-1120; Yamada et al. (1994)
Virology 205:121-126, and Dropulic et al. (1992) Journal of
Virology 66(3):1432-1441 provide an examples of HIV-1 specific
hairpin and hammerhead ribozymes.
[0130] Briefly, two types of ribozymes that are particularly useful
in this invention include the hairpin ribozyme and the hammerhead
ribozyme. The hammerhead ribozyme (see, Rossie et al. (1991)
Pharmac. Ther. 50:245-254; Forster and Symons (1987) Cell
48:211-220; Haseloff and Gerlach (1988) Nature 328:596-600; Walbot
and Bruening (1988) Nature 334:196; Haseloff and Gerlach (1988)
Nature 334:585; and Dropulic et al and Castanotto et al., and the
references cited therein, supra) and the hairpin ribozyme (see,
e.g., Hampel et al. (1990) Nucl. Acids Res. 18:299-304; Hempel et
al., (1990) European Patent Publication No. 0 360 257; U.S. Pat.
No. 5,254,678, issued Oct. 19, 1993; Wong-Staal et al.,
PCT/US94105700; Ojwang et al. (1993) Proc Natl Acad Sci USA
90:6340-6344; Yamada et al. (1994) Human Gene Therapy 1:39-45;
Leavitt et al. (1995) Proc Natl Acad Sci USA 92:699-703; Leavitt et
al. (1994) Human Gene Therapy 5:1151-1120; and Yamada et al. (1994)
Virology 205:121-126) are catalytic molecules having antisense and
endoribonucleotidase activity. Intracellular expression of
hammerhead ribozymes and a hairpin ribozymes directed against HIV
RNA has been shown to confer significant resistance to HIV
infection. These ribozymes are constructed to target a portion of
the HIV genome, or nucleic acid encoded by the genome. Preferred
target sites in HIV-1 include the U5 region, and the polymerase
gene. GUC and GUA cleaving trans active anti-HIV ribozymes are
known.
[0131] A decoy nucleic acid is a nucleic acid having a sequence
recognized by a regulatory nucleic acid binding protein (i.e., a
transcription factor, cell trafficking factor, etc.). Upon
expression, the transcription factor binds to the decoy nucleic
acid, rather than to its natural target in the genome. Useful decoy
nucleic acid sequences include any sequence to which a viral
transcription factor binds. For instance, the TAR sequence, to
which the tat protein binds, and the HIV RRE sequence (in
particular the SL II sequence), to which the rev proteins binds are
suitable sequences to use as decoy nucleic acids.
[0132] A transdominant nucleic acid is a nucleic acid which
expresses a protein whose phenotype, when supplied by
transcomplementation, will overcome the effect of the native form
of the protein. For example, tat and rev can be mutated to retain
the ability to bind to TAR and RRE, respectively, but to lack the
proper regulatory function of those proteins. In particular, rev
can be made transdominant by eliminating the leucine-rich domain
close to the C terminus which is essential for proper normal
regulation of transcription. Tat transdominant proteins can be
generated by mutations in the RNA binding/nuclear localization
domain. Reciprocal complementation of defective HIV molecular
clones is described, e.g., in Lori et al. (1992) Journal of
Virology 66(9) 5553-5560.
EXAMPLES
[0133] The following examples are provided by way of illustration
only and not by way of limitation. Those of skill will readily
recognize a variety of noncritical parameters which are changed or
modified to yield essentially similar results.
Example 1
Construction of FIV Packaging Plasmids and Vectors
[0134] The FIV long terminal repeat (LTR) is inactive, or minimally
active, in human cells. No prior method for high-level expression
of the full complement of proteins of a non-primate lentivirus in
trans in human cells and in replication-defective fashion has been
previously described. Therefore, to express high levels of FIV
proteins in trans in replication-defective fashion in human cells,
and to enhance safety by replacing a critical portion of FIV needed
for replication, FIV clone 34TF10 was cleaved with EspI, treated
with Klenow polymerase in the presence of 200 .mu.M dNTPs, then
cleaved with SacI, treated with T4 DNA polymerase in the presence
of 200 .mu.M dNTPs, and the resulting fragment containing the viral
coding regions (but not the LTRs) was gel purified. This fragment
was then blunt end-ligated into the NotI & XbaI cleaved,
Klenow-treated, calf intestinal alkaline phosphatase-treated
backbone of the CMV-expression plasmid pRc/CMV. The resulting
plasmid (CF1) was confirmed by multiple diagnostic restriction
digests. CF1 contains the CMV promoter followed by the FIV genome
from the distal portion of the post-LTR 5' leader (93 nt upstream
of the major splice donor) to 38 nucleotides downstream of the last
ORF (Rev) of FIV.
[0135] FIV clone 34TF10 was chosen for this work because this FIV
clone already has a mutation inactivating the ORF2 gene. See,
Talbott, R. L. et al. (1989) Proc. Natl. Acad. Sci. USA 86,
5743-5747. ORF2 is a putative transactivator, which has been shown
to be necessary for replication in feline peripheral blood
lymphocytes. Thus 34TF10 is an attenuated virus; this attenuation
is beneficial for this invention as it further minimizes the risk
of wild-type pathogenic FIV while not affecting the vector
performance. However, other clones with similar properties are
available or can easily be derived by similarly inactivating the
wild-type virus using recombinant methods.
[0136] Transfection of CF1 into human HeLa, 293 and 293-T kidney
cells by the calcium phosphate co-precipitation method produced the
surprising result of widespread ballooning syncytia
(multi-nucleated giant cells produced by fusion of
envelope-expressing cells with other cells) containing fifty to
several hundred nuclei and high levels (over one million cpm) of
supernatant reverse transcriptase. In fact, we found that human 293
and 293-T cell and HeLa monolayer cultures were reproducibly 90-95%
destroyed by syncytia after transient transfection of CF1 (for
example by transfection of 10/g of CF1 in a 75 cm.sup.2 flask), yet
as designed and expected, no infectious or replication-competent
virus is produced: transfer of large volumes of CF1-transfected
293-T cell supernatant to fresh 293 or 293-T cells or to Crandall
feline kidney cells resulted in no syncytia or RT production.
CF1.DELTA.env, which is identical to CF1 except for an FIV envelope
deletion (see, details below), does not cause syncytia but does
produce high levels of reverse transcriptase and of viral functions
needed for packaging of vectors. This unprecedented result produced
the novel insight that all the functions of FIV needed for protein
production, including the Rev/RRE axis of regulation, FIV gag/pol
production and envelope-mediated syncytia, can take place in human
cells if the FIV promoter is specifically replaced with a promoter
active in human cells.
[0137] The syncytia produced in human cells by CF1 and CT5 (below)
were completely inhibited by inclusion of a 1:1000 dilution of
plasma from FIV-infected cats (IC50 between 1:10,000 and 1:30,000
dilution) but were not at all inhibited by any dilution (even 1:10)
of plasma from uninfected domestic cats. This specific inhibition
by anti-FIV antibody provided further evidence that the syncytia
were FIV envelope-specific.
[0138] Radioimmunoprecipitation of 35S-methionine and
35S-cysteine-labeled human (293-T, HeLa) and feline (CRFK) cells
transfected with CF1 and the parental virus (34TF10) showed that
FIV viral proteins could be specifically immunoprecipitated with
FIV+sera from CRFK cells transfected with either plasmid. In the
human cells, 34TF10 produced little or no protein, indicating
minimal activity of the FIV promoter, while CF1 produced large
amounts of FIV protein. These proteins were shown to be FIV
specific by their absence in immunoprecipitates of the same cells
transfected with a control plasmid.
[0139] To allow heterologous (e.g., VSV-G) envelope use, specific
deletion of the FIV envelope (the product of the env gene) from CF1
was performed by a 3 part ligation: CF1 was restricted with PflmI
(which is present 4 times in CF1). Since the two PflM sites of
interest in the env gene were incompatible, and because simple
blunting of these PflM sites would produce an in-frame env
deletion, the PflM digest was treated with T4 polymerase to remove
the 3' PflmI overhang and ligated to a frame-shifting sacII linker,
digested with SacII, and gel purified. Aliquots of the
PflMI/SacII-linked digest were then restricted with sacII and
separately with either pvuI or Bsu36I. A 3-part ligation was then
performed (PvuI-BsU36I plus BsU36I-PflM(SacII linker) plus (SacII
linker)PflMI-PvuI). The resulting plasmid (CF1.DELTA.env), was
confirmed by multiple diagnostic restriction digests. CF1.DELTA.env
contained an 875 nt deletion in env. It produced high levels of
reverse transcriptase in human cells after transfection, but no
syncytia, providing further evidence, in addition to the plasma
inhibition experiments described above, that the syncytia seen with
CF1 were specifically FIV envelope-mediated.
[0140] The envelope function for CF1.DELTA.env can be supplied in
trans by any number of heterologous viral envelope proteins. These
include, but are not limited to, VSV-G, the amphotropic envelope of
Moloney murine leukemia virus (MoMuLV), and gibbon ape leukemia
virus (GALV) envelope, which are well known to those skilled in the
art for being able to efficiently supplant envelope function for
retroviruses.
[0141] In other embodiments, additional deletions of FIV sequences
are made from CF1.DELTA.env. For example, the region between the
major 5' splice donor and the gag ATG codon, although only twenty
nucleotides in FIV (compared to greater than 40 in HIV-1 and
greater than 70 in HIV-2), can be deleted or changed in sequence.
Additional env sequences are removed and tested and the effects of
deleting vif or other regions tested.
[0142] In a preferred embodiment, it was decided to completely
replace the promoter function of the FIV U3 in this system (that
is, in both packaging and vector constructs) for several reasons
(U3, or 3-prime unique region, contains the promoter/enhancer
elements of a retrovirus). First, the FIV LTR is inactive or poorly
active in human cells and it was desired to attempt production of a
vector in human cells since that could increase the likelihood of
subsequent human cell transduction and because good transfection
systems using feline cells are not well-characterized. Second, the
well-known high levels of expression directed by a promoter such as
the hCMV immediate early promoter in defined systems (e.g., the
293T human embryonic kidney cell system) are desirable. Third,
replacing U3 in both vector and packaging construct will further
help to eliminate the risk of replication-competent FIV. In another
modification 80 bases of the 3-prime U3 region was also deleted
from the vector, including especially the TATA box. Therefore,
replication-competent FIV cannot be regenerated because of this
deletion, and because the packaging plasmid has both the env gene
deletion and the ORF2-inactivating stop codon. Fourth, having all
three components (packaging plasmid, vector, envelope-expression
plasmid) driven from the same promoter can enhance synchronized
expression and efficient particle formation. Fifth, as described
above, human cell production is safer than feline cell production
for clinical use because feline cells pose the risk of introducing
known or unknown infectious agents into humans.
[0143] The design of the construct involves the fusion of the CMV
promoter and the FIV genome (from the 5' R repeat on) precisely at
the TATA box. First, PCR (synthetic PCRs were performed with
exonuclease+Vent polymerase) was performed with a SacI-tailed sense
PCR primer homologous to the nucleotides immediately downstream
from the FIV TATA box (5'-atataGAGCTCtgtgaaacttcgaggagtctc-3') in
combination with an antisense PCR primer
(5'-ccaatctcgcccctgtccattcccc-3') homologous to the opposite strand
of the FIV gag gene. The PCR product generated was 450 bp. This PCR
product was digested with XhoI before sac I digestion (because
there is a sac site is immediately following the XhoI site in the
FIV LTR and a SacI-SacI fragment would otherwise be generated).
After XhoI digestion and subsequent SacI digestion, the 310 bp
fragment resulting was:
[0144]
GAGCT/Ctgtgaaacttcgaggagtctctttgttgaggacttttgagttctcccttgaggctcccac-
agatacaataaatatttgagattgaaccctgtcgagtatctgtgtaatcttftttacctgtgaggtctcggaat-
ccgggccgagaacttcgcagttggcgcccgaacagggacttgattgagagtgattgaggaagtgaagctagagc-
aatagaaagctgttaagcagaactcctgctgacctaaatagggaagcagtagcagacgctgctaacagtgagta-
tctctagtgaagcggaC.backslash.TCGAG ctc.
[0145] This fragment was gel-purified away from the 140 bp residue
and cloned into the SacI-XhoI back-bone of the pRc/CMV plasmid. In
summary, this step arranges the FIV LTR sequences downstream of the
TATA box in precise register to both the TATA box of the CMV
promoter and the replaced TATA box of FIV: it places a SacI site 3
nucleotides downstream of the TATA box just as there is a SacI site
3 nucleotides downstream of the TATA box in the hCMV promoter and
it places the FIV R repeat precisely 9 nt downstream from the TATA
box just as in the FIV genome. The fusion therefore preserves (and
conjoins) the nucleotide spacing of both the CMV promoter and the
FIV transcriptional sequences and is detailed in the sequence
below:
[0146] . . .
acgtataagttgttccattgtaagagtaTATAAccagtgcttgtgaacttcgaggagtctc-
tttgttgagga FIV
AGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACC . . .
pRc/CMV
AGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCtgtgaaacttcgaggagtctcttt-
gttgazga CRF1
[0147] CRF1 completed the 5-prime fusion of the CMV promoter to the
FIV R repeat, but the 3-prime LTR and the remainder of the FIV
genome remained to be placed downstream. Therefore, sal I-tailed
PCR primers F3' S (tatataGTCgactagggactgtttacgaac) and F3' A/Not
(atatatagtcgacGCGGCCGCtgcg- aagttctcg) were used to amplify the 3'
FIV LTR: the SaLI-digested PCR product was ligated into the
alkaline phosphatase-treated compatible Xho I site of CRF1 and the
ligation was heat-inactivated and selected against wild-type with
Xho I. The resulting plasmid, named CRF(L), has both LTRs, and
places a unique NotI site at the 3' terminus of the 3' LTR.
[0148] Next, the major coding region of the FIV genome was inserted
by ligating the 8,845 nt BbeI-EspI fragment of p34TF10 into the
phosphatased BbeI-EspI backbone of CRF(L). The resulting plasmid
was called CT5 (for CMV promoter-->fusion at Tata
Box-->complete FIV genome from R repeat to normal proviral
terminus at the 3' LTR U5 element). CT5 encodes full-length,
infectious FIV which is promoted from the CMV promoter in the first
round (subsequent rounds is FIV LTR promoted since reverse
transcription generates a wild-type U3 at the 5' provirus
terminus). Transfection of either CT5 or the wild-type 34TF10 in
human 293-T cells resulted in both equivalent levels of RT in
supernatants and in widespread syncytia in a standard assay for FIV
replication: syncytia formation on 0.5% serum-maintained feline
Crandall feline kidney cells (CRFK cells) following transfer of
filtered supernatant from the transfected 293-T cells (See also,
Tozzini et al. (1992) Journal of Virological Methods 37, 241-252).
However, identically to 34TF10-generated virus, CT5-generated virus
is fully wild-type in its tropism: it does not replicate in human
cells. This is expected since the three-prime U3 of CT5 is wild
type (retroviruses carry their promoter at the three-prime end of
the virion mRNA: reverse transcription results in placement of a
copy of the three-prime U3 at the 5' end of the integrated provirus
where it acquires the ability to promote subsequent rounds of
transcription in susceptible cells). In summary, FIV that is
replication-competent for feline cells was produced by transfection
of CT5 into adherent cell lines, including 293T cells, proving that
the productive phase of the life cycle, but not replication, was
efficiently achieved in human cells.
[0149] FIV Vectors Derived From CT5.
[0150] Whether transfected into human or feline cells, CT5
generates FIV that is replication-competent for feline but not
human cells: the viral transcripts are expressed from a human, not
feline, promoter in producer cells, but the feline three-prime U3
remains intact and the source of the five-prime U3 promoter in any
subsequent round of replication is always the three-prime U3 of the
producer cell transcript. This remaining three-prime U3 element can
also be deleted.
[0151] To generate retroviral vectors from modifications of CT5,
two strategies were used. One embodiment takes note of the fact
that the RRE of FIV is located three-prime to coding sequences in
FIV (at the end of the TM protein rather than at the SU/TM junction
as in other lentiviruses). Therefore, if a reporter gene cassette
having its own polyadenylation p(A) signal is placed in antisense
orientation with respect to the FIV sequences, the position of the
RRE will be preserved enhancing utilization of cis-acting signals
and packaging. In a second embodiment, the RRE was removed from its
usual 3-prime location and placed by standard cloning techniques
immediately after a portion of the FIV gag/pol gene allowing both
Rev protection of the gag sequence-containing vector transcript and
insertion of a reporter gene cassette in sense orientation
downstream. The gag gene sequences are included in these vectors
because they have been shown to enhance packaging in other
retroviruses. However, in these vectors, the gag gene is
frameshifted by blunted closure of a Tth III 1 site located at
nucleotide 298 of gag (cloned by Tth III1 digestion, Klenow
polymerase-mediated filling, ligation, ligase inactivation, and
finally Tth III 1 selection against wild type) to prevent
recombination of functional gag and to prevent generation of a
transdominantly-supressive Gag protein. Additional portions of gag
beyond or even preceding this frameshift can be removed from
vectors without affecting titer.
[0152] Note that transcription of these vectors occurs from the CMV
promoter and not the FIV promoter. Other promoters are optionally
employed. In addition, multiple modifications are possible by
deleting additonal regions of the FIV genome, e.g., regions of gag,
as detailed below.
[0153] Such vectors are described and illustrated below:
[0154] 1. Vector CTAGCgfsB. The underlined, bold-faced letters in
the capsule descriptions, such as the one in parentheses here,
indicate the derivation of the name of the vector (CMV promoter
joined at TATA box driving expression of an internal reporter gene
cassette CMV-GFP-p(A) in reverse orientation and having a gag gene
frame shift mutation and subsequent insertion of the sv40 T antigen
binding site to cause plasmid amplification after transfection in
SV40 T antigen-expressing cells). CTAGCgfsB was constructed by
three-part ligation of the pvuI-EcoR1 fragment of CT5, the
EcoR1-Spe1 fragment of a green fluorescent protein (GFP)
gene-containing plasmid, pZcmvGFPpA, and the SpeI-PvuI fragment of
CT5. This ligation places the CMV promoter-GFP-p(A) signal cassette
in reverse orientation between EcoRI and SpeI in the FIV genome.
Next, the Tth III 1 site in the portion of gag/pol remaining in the
vector was cleaved, filled in with Klenow polymerase in the
presence of 200 .mu.M dNTPs and closed with T4 DNA ligase. This
blunting inserts an extra G residue which frameshifts the gag
fragment within a few bases of the Tth III 1 site. No pol sequences
are present. Therefore, the gag/pol precursor must be supplied in
trans from CF1.DELTA.env or subsequent modifications of
CF1.DELTA.env. Moreover, this step reduces the chance of wild-type
recombination and the chance that a transdominantly-interfering Gag
protein fragment will be produced. Finally the sv40-promoter-neoR
cassette from pRc/CMV was inserted in the plasmid outside of vector
sequences in order to benefit from Sv40 T antigen-driven plasmid
amplification and to allow selection for neoR if needed. In one
modification, the gag gene ATG start codon can be mutagenized to a
stop codon. Also the region between the BsRG1 site and upstream
PstI sites is optionally deleted to remove more of gag.
[0155] 2. Vector CTRZLb. (CMV promoter joined at TATA box driving
expression beginning with the R repeat of an internal
sense-oriented LacZ reporter gene cassette and having the 3' LTR
after lacZ; It also has a gag gene frame shift mutation at the Tth
III 1 site and subsequent insertion of the sv40 T antigen binding
site to cause plasmid amplification after transfection). To
construct this vector, CTAGCgfs was deleted between EcoR1 and BsrG1
by cleavage with these enzymes, Klenow treated with 200 .mu.M
dNTPs, blunted closure ligated with T4 ligase and selected against
wild-type with EcoR1. The BsrG1 site was regenerated. The
BsrgI-EcoNI fragment of pz-lacz was Klenowed and blunted into the
unique EcoNI site. The structure was thus: CMV promoter--Tata box
fusion with FIV R repeat--U5--.DELTA.gag--FIV Rev Response
element--cmv promoter-LacZ gene--LTR. Finally, the
sv40-promoter-neoR region from pRc/CMV was inserted in the plasmid
downstream of the vector to provide a T Antigen binding site in
order to benefit from Sv40 T antigen-driven plasmid amplification
and to allow selection for neoR if needed. The gag gene ATG start
codon can be mutagenized to a stop codon and additional portions of
gag are optionally deleted.
[0156] Vector supernatants generated by the calcium phosphate
co-transfection method were titered on human (HeLa, 293) and feline
(CRFK, Fc3Tg) cells. Compared to conventional Moloney murine
leukemia virus (Mo-MuLV)-based retroviral vectors the FIV vectors
were equally efficient at transducing human and feline cells.
Titers of 107 were achieved on both feline and human cells after a
single round of concentration by ultracentrifugation. Higher titers
are achievable with refinement of transfection and further
ultracentrifugation. In addition, both Human (HeLa) and feline
(CRFK) cells are efficiently transduced when growth-arrested using
aphidicolin 15 .mu.g/ml in the culture medium added 24 hours prior
to transduction and replaced daily through lacZ staining. LacZ
titers with the FIV vector in aphidicolin-arrested cells were
80-90% of those in dividing cells, while Mo-MuLV vectors
transduction was eliminated by aphidicolin treatment. These results
indicate that the FIV vector can transduce human cells and clearly
has lentivirus-specific biological properties lacking in
conventional (e.g., murine) retroviral vectors: the ability to
transduce non-dividing cells. Importantly, we detected no
preference of the FIV vectors for feline cells: the relative
transduction of various human and feline cells was cell-specific
not vector-specific. In other words, cells for which transduction
by the FIV vector were efficient were also efficiently transduced
by conventional Moloney murine leukemia virus vectors, and vice
versa.
[0157] This invention is applicable to human gene therapy. As
detailed above, these retroviral vectors have safety advantages
over HIV-based lentiviral vectors because HIV vectors are derived
from lethal human pathogens. Since FIV vectors can be worked with
in BL-2 level facilities, risks posed to personnel involved in
their production are lessened compared to HIV vectors, and ease and
convenience of production is enhanced. These vectors have
advantages over other gene delivery methods if stable gene transfer
to non-dividing or infrequently dividing cells is desired. Such
cells include, but are not limited to, cells of the human nervous
system, eye, hematopoietic system, integument, endocrine system,
hepatobiliary system, gastrointestinal tract, genitourinary tract,
bone, muscle, cardiovascular system and respiratory system. These
vectors also prevent exposure of patients to non-human cells and
prevent exposure to lentiviral genes or lentiviral proteins derived
from known pathogens.
EXAMPLE 2
FIV Based Lentiviral Vector Transduction of Non-Dividing Human
Cells and Demonstration of a CXCR4 Requirement for FIV Infection
and Cytopathicity.
[0158] HIV-based lentiviral vectors efficiently transduce
non-dividing cells, but are problematic because of their derivation
from lethal human pathogens. However, use of the non-primate
lentiviruses was complicated by a relative lack of knowledge about
their molecular properties, particularly their adaptability to
human cells. This example describes both productive and
post-receptor infective mechanisms of the FIV life cycle in human
cells and shows that the functions necessary for lentiviral vector
transduction can occur at high efficiency. Substitution of the FIV
promoter, which functioned poorly in non-feline cells was
performed. An entirely heterologously-promoted and env-pseudotyped
FIV vector system demonstrated high level human cell expression and
processing of FIV proteins in trans and produced FIV retroviral
vectors that transduced dividing, growth-arrested and post-mitotic
human cells (macrophages and hNT neurons) at high titer. The system
eliminates safety risks of feline producer cells. Severe
cytopathicity of heterologously promoted FIV envelope protein in
human cells was observed and vectors of the invention were used to
investigate this phenomenon. Expression of the FIV genome from the
human cytomegalovirus promoter induced profuse, Env-specific
syncytia in a wide variety of human cells but not in rodent cells.
Moreover, this fusogenic activity required co-expression of CXCR4,
the co-receptor for syncytium-inducing strains of HIV. However,
despite its CXCR4-dependence, non-host cell FIV Env syncytium
induction was dissociable from viral entry: CXCR4 expression in
non-feline cells permitted FIV Env-specific cell fusion but neither
FIV Env-mediated vector transduction nor viral replication.
Consistent with a co-receptor role, human CXCR4 expression in
feline cells changed viral phenotype from non-cytopathic to highly
cytopathic and increased both viral infectivity and transduction by
FIV-enveloped vectors. Utilization of CXCR4 by evolutionarily
distant lentiviruses implicates a fundamental role for this
chemokine receptor in lentiviral replication and cytopathicity. The
results have implications for comparative lentivirus biology as
well as for human gene therapy.
[0159] An ORF2-defective molecular clone (FIV 34TF10)19 of the
Petaluma strain was used (FIG. 1). At the top of FIG. 1, FIV 34TF10
and plasmid CF1 are shown. Further modifications of CF1 used in
this study are illustrated and described under the drawing for FIV
34TF10. To generate CF1, the Sac1-Esp1 fragment of 34TF10 was
blunt-end ligated between Not1 and Xba1 in the polylinker of the
hCMVIE promoter expression plasmid pRc/CMV (Invitrogen). Fusion of
hCMVIE promoter to FIV genome between TATA box (CMVIEp-derived) and
start of R repeat (FIV-derived) was performed and is shown for
plasmid CT5 (junction at the Sac1 site). The PCR-generated fusion
arranges the FIV R repeat sequences downstream of the CMV TATA box
in precise register to the replaced FIV TATA box; it also drives
exression of the vectors (bottom), which lack all vif, ORF2, pol
and env sequences. The marker gene cassette is in sense orientation
for lacZ and antisense orientation, with an additional poly(A)
signal, for GFP. A frameshift was introduced in the vectors at nt
298 of the remaining gag fragment. To generate pseudotyped vector,
the VSV-G expression plasmid pHCMV-G 42 (not illustrated) was
co-transfected in 293T cells with CF1.DELTA.env and the vectors
shown. Additional cloning details are found in Example 1.
[0160] 34TF10 productively infects Crandell feline kidney cells
(CRFK cells) but not feline peripheral blood lymphocytes or primary
feline macrophages (Carpenter & O'Brien (1995) Current Opinion
in Genetics and Development 5, 739-745; Waters et al. (1996)
Virology 215, 10-16; Sparger et al. (1994) Virology 205, 546-553;
Bandecchi et al. (1995) New Microbiologica 18, 241-252; Tozzini et
al. (1992) Journal of Virological Methods 37, 241-252 (1992);
Olmsted et al. (1989) Proceedings of the National Academy of
Sciences of the United States of America 86, 2448-2452). This
restricted tropism maps to the ORF2 mutation, not env: repair of
ORF2, a putative LTR transactivator, results in productive 34TF10
infection of all of these feline cell types36. The 34TF10 envelope
may thus be considered by loose analogy to HIV to be "dual-tropic;"
however, the lab adapted/T-tropic versus primary/Macrophage-tropic
classification has not been established for FIV strains or clones.
In fact, although CD4-depletion leading to AIDS is characteristic,
FIV also infects CD8.sup.+T cells and B cells as well as CD4.sup.+T
cells in infected felines (Pedersen (1993) supra). Neither 34TF10
nor any other domestic cat strains or clones are cytolytic in CRFK
cells; small multi-nucleated giant cells (4-12 nuclei) can be
detected in a maximally infected culture, but extensive cell death
does not occur (Barr et al. (1995), supra; Tozzini et al. (1992)
Journal of Virological Methods 37, 241-252; Barr (1997) Virology
228, 84-91). 34TF10 does not cause significant viremia or disease
in experimentally infected domestic cats; the in vivo phenotype of
the ORF2-repaired clone is not yet known (Sparger et al. (1994)
Virology 205, 546-553).
[0161] As diagrammed in FIG. 1, the human cytomegalovirus immediate
early gene promoter (hCMVIEp) was arranged either (a) to replace
all of the FIV LTR with a junction 97 nt upstream of the FIV major
5' splice donor (in plasmid CF1) or (b) to selectively replace the
FIV U3 promoter elements (in plasmid CT5, and in FIV vectors)
through a fusion at position -14 between the TATA box and the start
of transcription, i.e., the R repeat. The arrangement places the
CMV TATA box in precise register to the replaced FIV TATA box with
respect to the start of transcription (position -27). CF1 lacks
both LTRs except for the 89 nt portion of the three-prime U3 that
overlaps the rev ORF (FIV has no homologue to HIV nef), thus
deleting cis-acting sequences needed for replication and
integration (U3 promoter sequences, tRNA primer binding site, R
repeats, U5 elements). See also, Example 1 for details of the
cloning of the constructs described in this example. CF1.DELTA.env
has an additional 875 nt deletion in env which spans the SU-TM
junction and is also frameshifting: this plasmid, which was used
for packaging of pseudotyped vectors, is thus defective for ORF-2,
env, and the cis-acting retroviral elements noted. CT5, in
contrast, encodes fully wild-type, replication-competent 34TF10.
The system therefore eliminates the need for the feline promoter.
The function of ORF2 is not conclusively defined, but FIV
LTR-transactivating activity described for its gene product36 would
thus also be dispensable.
[0162] Transfection of CF1 or CT5 but not 34TF10 into human cells
resulted in explosive syncytium formation within 12-18 hours. HeLa
cell and 293 human embryonic kidney cell monolayers were
reproducibly 90-95% destroyed by syncytial lysis within 48-60 hours
after transfection of CF1 or CT5 (FIG. 1). All three plasmids
always produced many fewer (400 fold), smaller (4-12 nuclei)
syncytia and no discernible cytolysis in CRFK cells.
[0163] Transfections were by calcium phopshate preicpitation except
for U87MG and U87MG.CXCR4 cells, which were electroporated. In all
cases, transfected cells in this study were compared by
quantitative syncytial focus assay using cells simultaneously
transfected with the same calcium-phosphate precipitate. Cells were
stained by crystal violet or, for focal infectivity assays, by
immunoperoxidase staining with FIV-Petaluma sera and a secondary
horseradish peroxidase-conjugated goat antibody to feline IgG41.
All comparisons were controlled by co-transfection of an
hCMVIE-promoted GFP or LacZ reporter plasmid as 10% of the input
DNA; only experiments with transfection efficiencies varying <5%
between compared cell lines are reported. Where lysis was extensive
at 24 hours for CF1, comparative transfection efficiency by GFP
fluorescence was assayed in parallel wells in the presence of a
1:300 dilution of FIV-infected domestic cat plasma to inhibit
syncytium formation. All cells were ATCC lines propagated in 10%
bovine serum with antibiotics; CRFK cells were grown in L50 medium
as described. The ATCC no for CRFK is ATCCCCL94.
[0164] Consistent with previous studies (Barr et al. (1995) Journal
of Virology 69, 7371-7374) 34TF10 and CT5 transfection of CRFK
cells (ATCC CCL 94) established persistent infection with high
levels of RT (>5.times.10.sup.5 cpm/ml) by day 7-14, but minimal
cytopathic effect (6-12 +/-4 syncytia, 4-8 nuclei each, per 9.6
cm.sup.2 well of a six well plate). However, as expected CF1 did
not produce infectious virus: passage of filtered supernatant from
CF1-transfected human or feline cells to 10.sup.7 CRFK cells or to
10.sup.7 human cells (HeLa, 293, H9, Molt4, supT1, U937) produced
no syncytia or RT production; the adherent cell lines were also
negative by a previously described focal infectivity assay (FIA)
(Remington et al. (1991) Journal of Virology 65, 308-312),
sensitive to <5 infectious units per ml in 34TF10- or
CT5-infected CRFK cells.
[0165] Whether produced in human or CRFK cells, neither p34TF10-nor
CT5-generated FIV replicated in any human cells. Nevertheless, the
syncytia produced by CF1 and CT5 transfection into human and feline
cells were specifically caused by the FIV envelope protein, since
transfection of CF1.DELTA.env never produced syncytia in any cells,
but did yield comparably high levels of RT.
[0166] Mg2.sup.+ dependent reverse transcriptase was measured as
described previously (Willey et al. (1988) Journal of Virology 62,
139-147) 52 hours after transfection of indicated plasmids in CRFK
cells, HeLa, 293 and 293T cells. Extensive syncytial lysis was seen
in cells transfected with CT5, CF1, CF1.DELTA.pol and pHCMV-G. The
latter two plasmids were included as controls for cell lysis to
verify viral specificity. Supernatant from H9 cells infected 2
weeks earlier with HIV-1 and HIV-2 at m.o.i of 1.0 were also
assayed for comparison. In FIG. 2, each point is the mean of
triplicate measurements.+-.S.E. Radioimmunoprecipitation of
transfected cells with FIV (Petaluma strain)-infected domestic cat
plasma was performed. 293 cells, HeLa and CRFK cells were
transfected with the indicated plasmids by calcium phosphate
precipitation in 25 cm.sup.2 flasks. At 27 hours (293 cells) or 48
hours (HeLa and CRFK cells) after transfection, cells were
radiolabeled for five hours with .sup.35S-cysteine and
.sup.35S-methionine in cysteine- and methionine-free medium with
7.5% dialyzed fetal bovine serum after a one hour pre-incubation in
this medium without isotope. Lysates were pre-cleared with normal
cat serum and protein A sepharose, incubated overnight with 10
.mu.l of FIV-infected cat plasma, precipitated with protein A
separose, and electrophoresed with pre-stained markers in 10 or
12.5% SDS polyacrylamide gels. Lysates were derived from
approximately 15-25% the amount of cells in the other lanes because
of loss of cells to extensive syncytial lysis. Panel B shows
inhibition of CF1-induced syncytia in 293T and HeLa cells by
FIV-infected domestic cat plasma. Syncytia were scored at 48 hours
as foci with >8 nuclei by crystal violet staining of
methanol-fixed cells. Dilutions of either FIV+(squares, circles) or
FIV-(diamonds, triangles) plasma were added to cells at the time of
transfection in 12 well plates and again with change of medium 14
hours later.
[0167] Syncytia in CF1-transfected human cells were potently
suppressed by FIV Petamula strain-infected domestic cat plasma with
50% inhibition on 293T and HeLa cells at 1:32,000-fold and
1:12,700-fold dilution respectively, while pre-immune domestic cat
plasma had no effect on syncytium formation at any dilution, even
1:10 (FIG. 2B). Moreover, syncytium induction was abrogated by a
smaller, 539 nt (nt 7322-7861), non-frameshifting deletion confined
to the SU portion of env (sparing the TM domain and the upstream
proteolytic cleavage site; see CF1.DELTA.SU in FIG. 1) suggesting
that both the SU and TM domains of the envelope are required for
syncytium-induction. Transfection efficiencies determined by GFP
reporter co-transfection in parallel wells in the presence of 1:300
diluted antiserum for the experiments were .ltoreq.10%, showing
that syncytial lysis was mediated by fusion of non-transfected
cells with transfected cells.
[0168] Expression in human cells was then further examined by
reverse transcriptase (RT) assays and by immunoprecipitation of
radiolabeled cells with plasma from FIV-infected domestic cats.
hCMVIE-promoted constructs expressed high levels of
Mg2.sup.+-dependent RT in human cells (HeLa, 293, 293T), and were
also superior to the native LTR of p34TF10 in both feline (CRFK)
cells and human cells (FIG. 2A). In contrast, LTR-directed RT
expression by 34TF10 in human cells was minimal; protein expression
by the hCMVIEp-chimeric constructs also exceeded 34TF10 expression
in the feline cells by a factor of approximately six-fold. A
pol-deletion mutant (CF1.DELTA.pol) produced no RT (FIG. 2A) but
produced syncytia as extensively as CF1.
[0169] Immunoprecipitation of transfected, radiolabeled human and
feline cells demonstrated a wild-type 34TF10 pattern of expression
by CF1 or CT5 in human cells in amounts commensurate with the RT
assays. p34TF10 expression was nevertheless clearly detectable by
RIPA in HeLa cells, but at considerably lower levels than the
CMVIEp-chimerics, and was undetectable (even after prolonged film
exposure) in 293 or 293T cells. Consistent with this result, small
syncytia (4-6 nuclei, 11-14 syncytia.+-.4, n=4, per well of a six
well plate) were detectable in HeLa cells 48 hours after p34TF10
transfection. The frame-preserving SU deletion of CF1.DELTA.SU
resulted in the predicted truncated envelope precursor; it runs in
a poorly resolved smear with the shortened SU/gp100 cleavage
product. The plasma used did not precipitate the TM protein from
any cells. The other two env mutants abrogated immunoprecipitable
Env production and no envelope mutant formed syncytia in any cells
Consistent with the RT data, CF1 expression was also superior to
LTR-driven expression in CRFK cells.
[0170] Taken together, these data show that high level expression
of the full genomic repertoire of a non-primate lentivirus occurs
in human cells and that promoter substitution permits the
productive phase of FIV replication (including the Rev/RRE
regulatory axis, splicing, production of both Gag/Pol and Env
precursors, and correct proteolytic processing of each) to occur
with maximal efficiency in human cells and at higher levels than
seen with either the native promoter or the CMVIE promoter in CRFK
cells. Selective U3 replacement (in CT5 and for use in vectors
described below) and full LTR substitution (for protein production
in trans) were both effective.
[0171] To examine post-entry phases of the FIV life cycle in human
cells, CT5 was used as the starting point for constructing
retroviral vectors containing internally promoted marker gene
cassettes that replace pol, env and the accessory genes as well as
a portion of gag. A frameshift mutation was introduced in all
vectors at nt 298 of the remaining gag ORF by blunted closure of a
TthIII 1 site, generating a stop codon at nt 319 (FIG. 1). Vector
CTRZLb was co-transfected with CF1.DELTA.env and the VSV-G
expression plasmid pHCMV-G in 293T cells by calcium phosphate
co-precipitation. At 48-96 hours after transfection, supernatants
of the FIV vector and of a control VSV-G-pseudotyped Mu-MLV lacZ
vector were cleared, filtered (0.45 EM), titered on HeLa cells and
then re-titered by limiting dilution on a panel of feline and human
cell lines; the experiments were done with cells that were either
growing or arrested in G1/S with aphidicolin at 20 .mu.g/ml. High
titers (10.sup.6) equivalent to those of a conventional Moloney
murine leukemia virus retroviral vector were achievable with a
single round of concentration (Burns et al. (1993) Proceedings of
the National Academy of Sciences of the United States of America
90, 8033-8037) by ultracentrifugation. Similar to HIV vectors, the
FIV vector was minimally affected by cell cycle arrest, while the
Mu-MLV vector transduction was eliminated, thus demonstrating this
lentivirus-specific property of the FIV vector and transferablity
of this property to human cells. Equally importantly, when compared
in growing cells to the VSV-G psudotyped Moloney murine leukemia
virus lacZ vector, the FIV vector displayed no significant
preference for feline cells (compare titer ratios, see plot in
Table inset). When allowed to proliferate after transduction with
CTRZLb, large (100-400 cells) homogeneously lacZ-positive colonies
were generated, indicating stable, clonal maintenance of the
transgene. Although cell lines varied considerably in
transducibility as expected, these differences were equivalent for
the Moloney and FIV vectors: that is, they were cell-specific
rather than vector specific and reflects susceptibility to
VSV-G-mediated transduction. The chief block to the infective stage
of the FIV life cycle in non-feline cells is thus shown to be at
the level of virion entry rather than further downstream.
[0172] To further assess the ability of the FIV vectors to
transduce non-dividing human cells, we transduced post-mitotic
human cells, employing the two most developed, definitive human
tissue culture models: primary human macrophages and hNT neurons.
hNT neurons are irreversibly differentiated, polarized human
neurons derived from the NT2 teratocarcinoma cells by a six week
process employing retinoic acid and several mitotic inhibitors.
Third-replate hNT cells, employed here, are irreversibly
post-mitotic, remaining so a year after transplantation into the
brains of nude mice, morphologically resemble primary neurons,
express a plethora of neuron-specific markers, and grows clumps of
neurons that elaborate functional axons and dendrites. hNT neurons
transduced at an moi of 1.0. showed lacZ staining was visible both
in cell bodies and in processes. Primary human macrophages showed
high background lacZ staining and were therefore transduced with
GFP vector CTAGCb on day nine after isolation from peripheral blood
of normal donors. These cells were transduced at high titer by the
FIV vector but not by a GFP-transducing Mu-MLV vector.
[0173] The determinants of FIV encapsidation have received no
previous study. Lentiviral encapsidation signals are more complex
than those of the murine oncovirinae (Lever, (1996) Gene Therapy 3,
470-471). The LTRs were deleted from CF1.DELTA.env to prevent
packaging and remove sequences needed for reverse transcription and
integration; the 20 nucleotides between the major splice donor and
the gag gene, a putative contribution to lentiviral packaging, is
exceptionally short in FIV (20 nt compared to 44 nt in HIV-1, 75 nt
in HIV-2 and 375 nt in Mu-MLV). Scrambling and deletion of this
region may improve titer.
[0174] To test whether FIV structural gene-encoding sequences were
transferred to target cells by transduction with CF1-packaged
vector, 106 CRFK cells were transduced at m.o.i.=10 with DNAsed
CTRZLb vector, yielding 99% transduction. 1 .mu.g of genomic DNA
from these cells was negative by PCR for gag sequences, while
simultaneous amplification of the same amount of this DNA spiked
with genomic DNA from as few as 10 cells from a chronically
34TF10-infected CRFK culture was positive.
[0175] Severe cytopathicity that resulted when FIV envelope
expression was enabled in human cells. Because of the recent
discovery of CXCR4 as the co-receptor for syncytium-inducing
strains of HIV, these observations raised immediate questions of
specific mechanism. The FIV primary receptor remains unknown. Since
most human cell lines, including HeLa and 293 cells, express
appreciable levels of CXCR4, we next employed those rare human
lines known to express no CXCR4 (U87MG and SK-N-MC cells) or
extremely low levels of CXCR4 (HOS cells) Berson et al. (1996)
Journal of Virology 70, 6288-6295; Endres et al. (1996) Cell 87,
745-756; Harouse & Gonzalez-Scarano (1996) Journal of Virology
70, 7290-7294). Transfection of CF1 or CT5 into U87MG and SK-N-MC
cells (by electroporation or calcium phosphate co-precipitation)
never resulted in syncytium formation (n=9 for each line,
transfection efficiencies.gtoreq.10% by GFP reporter
co-transfection). In addition, while CF1-transfected rat 208F cells
readily fused with a variety of human cell lines and with CRFK
cells, fusion did not occur with SK-N-MC or U87MG cells. In
addition, transfection of other rodent cells (NIH 3T3, CHO) cells
did not produce syncytia (n=8, efficiencies.gtoreq.10% by GFP
co-transfection).
[0176] We therefore constructed an MMLV-based retroviral vector,
pZ.CXCR4, that expresses human CXCR4 and neoR from a bicistronic
mRNA and used it to generate a panel of G418-selected U87MG, HOS,
SK-N-MC, NIH3T3 and CRFK cell lines expressing human CXCR4. Control
G418-selected lines were generated with the parental retroviral
vector, pJZ30854. Expression of CXCR4 was documented in all
pZ.CXCR4-transduced lines. Transfection of CF1 or CT5 into
U87MG.CXCR4, SK-N-MC.CXCR4, 3T3.CXCR4, but not simultaneous
transfection into the respective control lines (n=8 for CF1, n=6
for CT5), produced exuberant syncytia. Moreover, while small 4-8
cell syncytia could be consistently observed in CF1-transfected HOS
cells, CF1-transfected HOS.CXCR4 cells produced massive
multinucleated giant cells containing several hundred nuclei. To
confirm these results, we also carried out feline/human cell mixing
studies with 3201-FIV cells (chronically FIV-infected feline cat
lymphoma cells, ATCC CRL 10909). U87MG, SK-N-MC, HOS, 3T3 cells,
their respective pZ.CXCR4 vector-selected counterpart lines, and
HeLa cells, were each plated (3.times.10.sup.5 cells) in six well
plates. 105 3201-FIV cells per well were added the next day. At 18
hours, large ballooning syncytia (involving 30-80% of the
monolayer) of the HeLa cells and each CXCR4-expressing line were
observed; no syncytia at all were seen at any time point in U87MG,
SK-N-MC, HOS, or 3T3 cells.
[0177] Transduction by FIV Env was then examined using
co-transfection of CF1 and CTRZLb in 293T cells. As shown in Table
1, this vector was unable to transduce any human lines, regardless
of CXCR4 expression; transduction could be detected in CRFK cells,
but at very low titer (<10 transducing units/ml). CRFK CXCR4
cells, in contrast, were transducible at least 2 logs higher titer
(3.6.times.10.sup.2) than CRFK. Moreover, the ligand for CXCR4
inhibited vector transduction via FIV envelope.
[0178] Since the vector data indicated that CXCR4 increased feline
cell viral entry as well as more broadly mediating fusogenesis, we
next compared the infectivity of replication-competent FIV on CRFK
cells and CRFK.CXCR4 cells. Each line was seeded into 48 well
plates at 10.sup.4 cells per well and infected the next day with
serial four-fold dilutions of a 34TF10 virus stock produced in CRFK
cells. To reduce artifactual loss of titer from the marked
cytopathicity of 34TF10 in CRFK.CXCR4 cells, the plates were fixed
and examined at 40 hours after infection by a focal infectivity
assay (employing 1:500 dilution of the FIV-positive sera and a
secondary horseradish peroxidase-conjugated anti-feline IgG
antibody) sensitive to 1 in 106 infected cells (see also, Remington
et al. (1991) Journal of Virology 65, 308-312 and Chesebro &
Wehrly (1988) Journal of Virology 62, 3779-3788). 34TF10 was eight
fold more infectious on CRFK.CXCR4 than on CRFK (p=0.0002). We
consider this a minimum estimate of infectivity ratio, since
numerous rounded floating or poorly adherent cells that stained by
IFA were present in the infected CRFK.CXCR4 wells as early as 40
hours (but not in uninfected control wells or infected CRFK wells)
and were not counted. To confirm these results, limiting dilution
titration of 34TF10 on the two cell lines was carried out by
infecting as above; cells were split at four-five day intervals for
three weeks and positive wells were identified by RT assay.
[0179] Taken together these results show that fusogenic activity
and viral entry of distantly related primate and non-primate
lentiviruses are mediated by the same cell surface molecule. This
broad utilization of CXCR4, for a non-primate lentivirus that does
not utilize CD4 as a primary receptor, implicates a fundamental
role for CXCR4 in lentiviral syncytiagenesis and in AIDS
pathogenesis. Our experiments do not exclude that low-level FIV
envelope-mediated infection of human cells occurs, perhaps via
CXCR4 alone, as has been well-described for some isolates of HIV-1
and HIV-2 (Endres et al. (1996) Cell 87, 745-756; Harouse &
Gonzalez-Scarano (1996) Journal of Virology 70, 7290-7294; McKnight
et al. (1994) Virology 201, 8-18; Reeves et al. (1997) Virology
231, 130-134; Potempa et al. (1997) Journal of Virology 71,
4419-4424; Harouse et al. (1995) Journal of Virology 69, 7383-7390;
Talbot et al. (1995) Journal of Virology 69, 3399-3406; Tateno et
al. (1989) Proceedings of the National Academy of Sciences of the
United States of America 86, 4287-4290 (1989). Clapham et al.
(1992) Journal of Virology 66, 3531-3537) but indicate that, as in
most of these examples, such a process is inefficient. Similarly,
rabbits can also be infected by HIV-1, but the process is quite
inefficient (Gardner & Luciw (1989) Faseb Journal 3,
2593-2606). Our data are consistent with the existence of a primary
FIV receptor. In addition to the vector data, for example, HeLa
cells do not support productive FIV replication but were easily
transducible with the VSV-G pseudotypes, expressed abundant CXCR4,
exhibited extensive Env-specific cytopathicity, showed low but
significant levels of FIV LTR-directed expression, permitted normal
viral protein processing, and generated replication-competent FIV
from plasmid CT5.
[0180] The results therefore have important implications for
lentivirus biology and for human gene therapy. By showing that a
non-primate lentivirus also utilizes CXCR-4 for both cell fusion
and viral entry, our data show that this chemokine receptor plays a
broadly fundamental role in lentivirus replication, in
syncytiagenesis, and perhaps in pathogenesis. It is also likely
that in vivo replication of FIV has additional and more subtle
requirements. FIV vectors therefore represent an inherently safer
alternative to HIV vectors. Epidemiologic support for this
hypothesis is strongest for FIV than for any other non-primate
lentivirus, because FIV has shown no ability to infect or cause
disease in humans after natural inoculation in many humans over
many years by the same principal infective route operative in cats
(cat bites). Furthermore, in addition to U3, ORF2 and env,
additional deletions of FIV sequences from both vectors and
packaging plasmids are possible. FIV vectors are logistically
easier to produce since infectious FIV is routinely propagated in
Biosafety Level 2 tissue culture (vectors herein are approved for
BL-2 use by the UC San Diego Institutional Biosafety
Committee).
[0181] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference in its
entirety for all purposes. Although the foregoing invention has
been described in some detail by way of illustration and example
for purposes of clarity of understanding, it will be readily
apparent to those of ordinary skill in the art in light of the
teachings of this invention that certain changes and modifications
may be made thereto without departing from the spirit or scope of
the appended claims.
* * * * *