U.S. patent application number 10/645451 was filed with the patent office on 2004-09-09 for hiv and cd4 transgenic animals and uses therefor.
Invention is credited to Bryant, Joseph L., Davis, Harry G. JR., Reid, William C..
Application Number | 20040177393 10/645451 |
Document ID | / |
Family ID | 24751403 |
Filed Date | 2004-09-09 |
United States Patent
Application |
20040177393 |
Kind Code |
A1 |
Bryant, Joseph L. ; et
al. |
September 9, 2004 |
HIV and CD4 transgenic animals and uses therefor
Abstract
The invention provides transgenic animals comprising a
lentiviral transgene, such as an HIV transgene. Also within the
scope of the invention are cells and eggs from the transgenic
animal. Further included are methods for identifying therapeutic
compounds for preventing lentiviral infection and treating
associated disease (e.g. AIDS).
Inventors: |
Bryant, Joseph L.;
(Rockville, MD) ; Reid, William C.; (Frederick,
MD) ; Davis, Harry G. JR.; (Woodbine, MD) |
Correspondence
Address: |
INTELLECTUAL PROPERTY / TECHNOLOGY LAW
PO BOX 14329
RESEARCH TRIANGLE PARK
NC
27709
US
|
Family ID: |
24751403 |
Appl. No.: |
10/645451 |
Filed: |
August 21, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10645451 |
Aug 21, 2003 |
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09685256 |
Oct 10, 2000 |
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6660904 |
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09685256 |
Oct 10, 2000 |
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PCT/US99/07821 |
Apr 9, 1999 |
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PCT/US99/07821 |
Apr 9, 1999 |
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09058113 |
Apr 9, 1998 |
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6156952 |
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Current U.S.
Class: |
800/14 ;
435/353 |
Current CPC
Class: |
A01K 2217/05 20130101;
C07K 14/005 20130101; A01K 2267/0337 20130101; C07K 14/70514
20130101; A01K 2227/105 20130101; C12N 15/8509 20130101; A01K
67/0278 20130101; A01K 2217/20 20130101; A01K 67/0275 20130101;
A01K 2217/00 20130101; C12N 2740/16122 20130101; C12N 2740/16022
20130101; A01K 2207/15 20130101 |
Class at
Publication: |
800/014 ;
435/353 |
International
Class: |
A01K 067/027; C12N
005/06 |
Claims
1. A transgenic rat, whose genome comprises at least one copy of a
transgene encoding at least a portion of a CD4 protein sufficient
for binding to gp120, wherein CD4 encoded by the transgene is
expressed on PMBCs of the transgenic rat.
2. The transgenic rat of claim 1, wherein the genome further
comprises a transgene encoding for at least a portion of CCR5.
3. The transgenic rat of claim 2, wherein CD4 is human CD4.
4. The transgenic rat of claim 3, wherein the human CD4 is a full
length CD4 protein.
5. The transgenic rat of claim 2, wherein the encoded transgene is
capable of mediating entry of HIV.
6. A cell from the transgenic rat of claim 3.
7. A cell from the transgenic rat of claim 4.
8. The cell of claim 6, which is a germ cell.
9. The cell of claim 8, which is a somatic cell.
10. The cell of claim 6, which is an egg.
11. A transgenic rat, whose genome comprises a gene encoding CXCR4
and at least one copy of a transgene encoding at least a portion of
a CD4 protein sufficient for binding to gp120, wherein CXCR4
encoded by the gene and CD4 encoded by the transgene is expressed
on PMBCs of the transgenic rat.
12. An assay for identifying a molecular antagonist compound, which
interferes with a lentivirus ligand-CD4 receptor interaction,
comprising the steps of: (a) administrating the molecular
antagonist compound to a transgenic rat of claim 1; and (b)
determining the level of interaction between the lentivirus ligand
and CD4 receptor expressed on PMBCs of the transgenic rat, wherein
a difference in the level of interaction between the lentivirus
ligand and CD4 receptor expressed on PMBCs of the transgenic rat
relative to that in a transgenic rat to which the compound was not
administered indicates that the compound interferes with the
lentivirus ligand and the CD4 receptor.
13. The method according to claim 12, wherein the lentivirus ligand
is HIV-1 gp120.
14. A method for identifying a compound which inhibits infection of
a human cell by HIV, comprising (a) administrating a test compound
to a transgenic rat of claim 3 or contacting a cell thereof with
the test compound; and (b) determining the level of IV or gene
product thereof in the transgenic rat or cell thereof of step (a),
wherein a lower level of HIV or gene product thereof in the
transgenic rat or cell thereof of step (a) relative to that in a
transgenic rat to which the test compound was not administered or
cell that was not contacted with the test compound, respectively,
indicates that the test compound inhibits infection by HIV.
15. The method of claim 14, comprising determining the level of HIV
by determining the level of p24.
16. The method of claim 14, comprising determining the level of one
or more HIV RNAs.
17. The method of claim 14, comprising determining the level of one
or more HIV proteins.
18. A method for identifying a compound which reduces infection of
a human cell by HIV, comprising (a) administrating a test compound
to a transgenic rat of claim 1; and (b) determining the presence of
at least one symptom characteristic of AIDS in the transgenic rat,
wherein the reduction of at least one symptom of HIV in the
transgenic rat of step (a) relative to that in a transgenic rat to
which the test compound was not administered, indicates that the
test compound inhibits infection by HIV.
19. A method for testing or determining the efficiency of a test
vaccine against HIV, comprising administering to a transgenic rat
of claim 1, a vaccine, infecting the transgenic rat with HIV, and
determining the level of HIV or gene product thereof, wherein the
presence of less HIV or product thereof in a rat having been
administered the vaccine relative to a transgenic rat that has not
received the vaccine indicates that the test vaccine is
efficient.
20. An assay for identifying a test compound, which interferes with
a lentivirus ligand-CD4 receptor interaction, comprising the steps
of: (a) incubating a cell of the transgenic rat of claim 3 with a
lentivirus ligand and the test compound; and (b) determining the
level of interaction between the lentivirus ligand and CD4 receptor
expressed on the cell of the transgenic rat, wherein a difference
in the level of interaction relative to that of a cell that was not
contacted with the test compound indicates that the test compound
interferes with the lentivirus ligand and the CD4 receptor.
21. The method according to claim 19, wherein the lentivirus ligand
is HIV-1 gp120.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of copending
U.S. patent application Ser. No. 09/685,256, filed Oct. 10, 2000,
which is a continuation-in part application of International
Application No. PCT/US99/07821, filed Apr. 9, 1999, which is a
continuation-in-part of application Ser. No. 09/058,113, filed Apr.
9, 1998. The contents of each of these applications are
specifically incorporated by reference herein.
BACKGROUND OF THE INVENTION
[0002] Human immunodeficiency virus (HIV) is an etiological agent
of Acquired Immune Deficiency Syndrome (AIDS). AIDS was first
reported in the United States of America in 1981. As of January
1997, approximately 1.5 million cases of AIDS in adults and
children had been reported to the World Health Organization (WHO);
however, because reporting is difficult, WHO estimates that there
were more than 8.4 million cases, about 580,000 of which reside in
the United States. However, the number of HIV infected individuals
is much higher: as of January 1997, WHO estimated that there were
approximately 29.4 million HIV infected individuals world-wide,
with about 1 million in the United States. It has been estimated
that by the year 2000, between 40 and 100 million individuals will
be infected with HIV.
[0003] HIV, which has also been referred to as
lymphadenopathy-associated virus (LAV), HTLV-III, or AIDS related
virus (ARV), is a lentivirus from the family of retroviruses and is
composed of RNA consisting of about 9,700 base pairs, three gag
proteins (having molecular weights of 55,000, 24,000 and 17,000
daltons), a reverse transcriptase (molecular weights of 66,000 and
51,000 daltons have been detected), three glycoproteins (two
molecules having molecular weights of 120,000 and 41,000 daltons,
and their precursor, a molecule with a molecular weight of 160,000
daltons, hereinafter abbreviated as gp120, gp41 and gp160,
respectively) which comprise the envelope, and other components.
Exposed, envelope proteins are particularly important for viral
infection and therefor also, the prevention thereof. As a result of
proteolysis, gp160 is cleaved into gp120 and gp41. Gp41 is a
transmembrane protein, which is incorporated into the lipid bilayer
of the viral envelope, while gp120 is exposed on the outside of the
envelope and some of it is released from the virus. Both gp41 and
gp120 possess many sugar-binding sites, and about half of the gp120
molecule is comprised of sugars. The gp120 molecule binds to the
CD4 antigens on the surface of cells, in particular helper T cells.
Once HIV is bound to CD4 via gp120, another env gene product, gp41,
mediates fusion between the membranes of the cell and the virus
allowing the core of the virus to enter the cell. Gp120, which is
expressed on the plasma membrane of infected cells before virus is
released, can bind to CD4 on another cell, initiating a membrane
fusion event resulting in syncytia formation, and HIV genomes can
be passed between the fused cells directly.
[0004] The env gene (gp 120) is the primary determinant of cell
tropism for both HIV and Simian Immunodeficiency Virus (SIV).
Variable region 3 (V3) of gp120 is a key component within env that
determines cell tropism. The efficiency of replication and the
ability to induce the syncytia formation are also affected by
changes in the V3 loop.
[0005] The first HIV virus isolated is referred to as HIV-1 and is
generally described in several articles, e.g., Barre-Sinoussi et
al., Science 220:868, 1983; Gallo et al., Science 224:500, 1984;
Popovic et al., Science 224:497, 1984; and Levy et al., Science
225:840, 1984, each of which is hereby incorporated by reference.
Various isolates of HIV-1 have been obtained from North America,
Western Europe and Central Africa. These isolates differ somewhat
in their nucleotide sequence, but the proteins they encode are
generally antigenically cross-reactive.
[0006] A second virus related to HIV-1 has been isolated and termed
HIV-2 (Guyader et al., Nature 326:662, 1987; Brun-Vezinet et al.,
The Lancet 1:128, 1987; and Clavel et al., Science 233:343, 1986).
The genetic organization of HIV-2 is similar to that of HIV-1. Of
the two distinct subtypes, HIV-1 is predominant and found
throughout the world, whereas HIV-2 has been isolated primarily in
West African countries such as Guinea Bissay, Ivory Coast, and
Senegal, with some cases also identified in the Americas and
western Europe. Epidemiological studies suggest that the incubation
period for HIV-2 for the development of disease is longer than for
HIV-1.
[0007] HIV isolates from around the world were found to differ in
nucleotide sequence. These sequences have been collected in a
specialized database (Myers et al. (1994) Los Alamos National
Laboratory, Los Alamos, N.M.). Two major groups of HIV have been
identified. Viruses of group M (for "main") are responsible for the
majority of infections worldwide; group O (for "outgroup") is a
relatively rare group currently found in Cameroon, Gabon, and
France. Group M can be divided into at least eight distinct
subtypes or clades (A through H) (Myers, supra; Louwagie et al.
(1995) J. Virol. 69:263). Isolates from HIV-1 from different clades
may differ by 30-40% in the amino acid sequence of the gp120 SU
protein; isolates within a clade vary from 5% to 20%. Clade B
predominates in North America and Europe and lade E predominates in
northern Thailand. Similarly, there are five HIV-2 sequence
subtypes.
[0008] A group of viruses isolated from monkeys, termed simian
immunodeficiency virus (SIV or STLV-III), is related to HIV-1 and
HIV-2, particularly the latter. See Daniel et al., Science
228:1201-1204 (1985); Kanki et al., Science 230:951-954 (1985);
Chakrabarti et al., Nature 328:543-547 (1987); and Ohta et al.,
Int'l. J. Cancer 41:115-222 (1988). Members of this viral group
exhibit minor variations in their genomic sequences, and have some
differences in their restriction enzyme maps.
[0009] Although human CD4 is essential for HIV infection, it is not
sufficient. Expression of human CD4 on rodent cells renders them
capable of binding virus but still nonpermissive for fusion or
infection (Maddon et al. (1986) Cell 47:333). The host component or
coreceptors, sometimes referred to as the "fusion receptors," were
identified only recently. These coreceptors are receptors for
chemokines (i.e. small proteins which serve as chemoattractants in
inflammation) and they permit HIV infection of virtually any
mammalian or avian cell that expresses human CD4 (Bates (1996) Cell
86:1-3). The most important coreceptors are CXCR4 (also called
"fusin" or "LESTR) (Endres et al. (1996) Cell 87:745; Feng et al.
(1996) Science 272:872) and CCR5 (Akhatib et al. (1996) Science
272:1955; Choe et al. (1996) Cell 85:1135; Deng et al. (1996)
Nature 381:661; Doranz et al. (1996) Cell 85:1149; and Dragic et
al. (1996) Nature 381:667). CXCR4 is the receptor for the chemokine
SDF-1, whereas CCR5 serves as a receptor for the chemokines MIP-1
and RANTES. These coreceptors play a crucial function for viral
entry into cells, and they are also the principal determinants of
tropism among CD4+ cells.
[0010] Two distinct types of HIV-1 have been identified based on
the cells in which they replicate in vitro. Viruses that replicate
in T cell lines, but not macrophages or monocytes, are referred to
as T tropic, whereas viruses with the complementary specificity are
referred to as M tropic. The tropism is at least a function of the
coreceptor: M tropic viruses can use only CCR5 for entry, and T
tropic viruses use CXCR4. A few dual tropic isolates capable of
using both are also known. T tropic viruses often cause infected
cells to fuse with uninfected cells if the latter express both
human CD4 and CXCR4. Such viruses are referred to as
"syncytium-inducing" (SI). All isolates can infect activated T
cells freshly isolated from peripheral blood, which are present in
PBMC cultures, since such cells express both CXCR4 and CCR5.
Furthermore, cell tropisms are not fixed and can change when the
virus is passaged in cell culture (Metlzer et al. (1990) Immunology
Today 11:217; Levy (1993) Microbiol. Rev. 57:183).
[0011] Two animal species (i.e., man and chimpanzee) are known to
be susceptible to HIV infection, but only in man does the disease
develop. HIV-1 transgenic mice carrying intact copies of the HIV-1
provirus have been obtained (Leonard et al. (1988) Science
242:1665). These mice develop a spontaneous and fatal disease that
mimics some of the features described in human AIDS. Other HIV-1
transgenic mice carrying the HIV-1 proviral DNA in which deletions
have been introduced have also been produced (see, e.g., Dickie et
al. (1991) Virology 185:109; Santoro et al. (1994) Virol.
201:147).
[0012] However, none of these transgenic mice closely model the
development of AIDS in humans. In particular, none of the HIV
transgenic mice express gp120 on the surface of their T cells.
Thus, syncytium formation between HIV infected cells and CD4+
cells, e.g., T cells, which is reported to occur in humans and
which is in fact the mechanism by which HIV is transmitted from one
cell to another without the production of infectious HIV particles,
does not occur in HIV transgenic mice. In addition, since HIV
transgenic mice do not express gp120 on the surface of infected
cells and all of the neutralizing antibodies in humans have mapped
to the envelope protein, gp160, or one of its component parts
(gp120 or gp41), transgenic, HIV mice are not particularly useful
for developing human HIV vaccines.
[0013] Thus, there is a need for animal models of AIDS and other
lentiviral diseases, which more closely model infection and disease
progression as it occurs in humans.
SUMMARY OF THE INVENTION
[0014] In one aspect, the present invention features non-human
animal models of lentiviral (e.g., HIV) infection and development
of disease (e.g. AIDS). Preferred non-human animals comprised of a
lentiviral transgene are larger than a mouse. Other preferred
non-human, transgenic animals are smaller than a monkey. A
particularly preferred non-human, transgenic animal is a transgenic
rat.
[0015] In preferred embodiments, the transgene comprises
essentially all of a viral genome, i.e., the transgene comprises at
least about 70%, at least about 80%, at least about 90% or at least
about 95% of a wild-type viral genome. Also within the scope of the
invention are transgenic animals in which the transgene comprises a
smaller portion of the wildtype virus, (e.g. less than about 70% of
the viral genome). For example, the transgenic animal can comprise
a transgene encoding a single protein.
[0016] In a preferred embodiment, the transgenic, non-human animal
is comprised of an HIV transgene. Exemplary HIV proteins for
inclusion in the transgene include an envelope protein (e.g., gp120
and gp40), a reverse transcriptase, a protease, an integrase, a
ribonuclease, a nucleocapsid core factor (gag), a transcriptional
activator (e.g., tat and vpr) or proteins encoded by the genes vif,
vpu, and nef.
[0017] In another preferred embodiment, the transgenic, non-human
animal expresses the HIV proteins. In a particularly preferred
embodiment, the animal expresses the HIV protein gp120 on the
surface of its peripheral blood mononuclear cells (PBMCs). In a
further preferred embodiment, the transgenic, non-human animal
expresses at least one HIV coreceptor (e.g. CCR5 or CXCR4).
[0018] In another embodiment, in addition to the lentiviral
transgene, the transgenic, non-human animal is comprised of at
least one additional transgene. In a preferred embodiment, the
additional transgene is a human CD4 receptor gene. Expression by
the animal of both HIV and human CD4 allows the HIV particles
produced to enter the CD4 expressing cells, thus resulting in HIV
infection. Other preferred animals of the invention include those
that are transgenic for human CD4, and which can, optionally, be
infected by a lentivirus, e.g., HIV. In another preferred
embodiment, the additional transgene is an HIV coreceptor, such as
CCR5 or CXCR4, which further aids in HIV infection. In a further
preferred embodiment, the additional transgene is a gene involved
in a disease or condition that is associated with AIDS (e.g.
hypertension, Kaposi's sarcoma, cachexia, etc.).
[0019] In another preferred embodiment, the non-human animal
containing an HIV transgene exhibits at least one symptom or
phenotype characteristic of human HIV infection and/or development
of AIDS (e.g. development of cataracts, cachexia or lesions (e.g.
skin lesions, for example, resulting from psoratic dermatitis,
hyperkerstotic lesions, kidney sclerotic lesions or inflammatory
lesions of the central nervous system).
[0020] In another aspect, the invention features methods for
producing the transgenic animals described herein. A preferred
method comprises the steps of: (a) obtaining a non-human animal egg
containing a lentivirus transgene; (b) implanting the egg of step
(a) into a female non-human animal; (c) selecting offspring
containing the transgene to thereby obtain a founder animal; and
(d) crossing the founder animal within the same species, but
opposite sex, to thereby produce a transgenic animal comprising a
lentivirus transgene.
[0021] In one embodiment, the lentiviral transgene, in step (a), is
an HIV transgene. In a preferred embodiment, the HIV transgene is
infectious. For example, the transgene can be supplied by a
wild-type HIV provirus, e.g, an HIV-1 or HIV-2 provirus or strain
thereof. Alternatively, the transgene can be a modified form of a
wild-type provirus, such as a provirus having a deletion,
substitution or addition of at least one nucleotide to the viral
genome. For example, a provirus can be modified by replacing the
transcriptional control element in an LTR of the HIV provirus with
another transcriptional control element, for example to alter the
tropism of the virus. A provirus can also be modified by deleting a
portion of, or mutating, at least one HIV gene, to thereby
inactivate at least one HIV protein, e.g, gag, pol, env, or tat. In
another embodiment, the HIV transgene is non-infectious. For
example, an HIV provirus can be rendered non-infectious by deleting
a portion of gag and pol or by mutating at least one LTR of the
provirus.
[0022] In a further aspect, the invention features non-human animal
cells containing a lentivirus transgene, e.g., an HIV transgene.
For example, the animal cell (e.g. somatic cell or germ cell (i.e.
egg or sperm)) can be obtained from a lentivirus transgenic animal.
Transgenic somatic cells or cell lines can be used, for example, in
drug screening assays. Transgenic germ cells, on the other hand,
can be used in generating transgenic progeny, as described
above.
[0023] In yet further aspects, the invention features methods for
using the non-human transgenic animals, cells and cell lines of the
invention for investigating molecular and cellular mechanisms of
lentiviral mediated pathogenesis (e.g. the molecular and cellular
mechanisms of the skin lesions, CNS disturbances, heart and kidney
disease, which is associated with human HIV infection); as well as
for identifying compounds and vaccines for treating and/or
preventing lentivirus (e.g. HIV) infection and disease (e.g. AIDS)
development.
[0024] A preferred in vitro assay for identifying molecular
antagonists which, for example, interfere with a lentivirus
ligand-receptor interaction, as well as molecular agonist which,
for example, function by activating a lentivirus protein (e.g.
receptor) is comprised of the steps of: (a) incubating transgenic
cells expressing a protein (e.g. receptor) known to be involved in
lentivirus infection with a test compound; and (b) detecting the
interaction between the lentivirus protein and the test compound,
wherein the presence of an interaction indicates that the test
compound may be an inhibitor of lentivirus infection. In another
embodiment, in step (a), the test compound is incubated with the
transgenic cell in the presence of a compound, which is a binding
partner (e.g. a receptor ligand) to the expressed protein and the
interaction between the test compound and the lentivirus protein or
between the lentivirus binding partner and the lentivirus protein
is detected.
[0025] In other embodiments, cell based assays can be used to
identify compounds which modulate expression of a lentivirus gene,
modulate translation of a lentivirus mRNA, or which modulate the
stability of a lentivirus mRNA or protein. A preferred assay
comprises the steps of: (a) incubating a transgenic cell, which
expresses a particular lentivirus protein with a test compound; and
(b) comparing the amount of the lentivirus protein produced to that
produced by the same cell which has not been contacted with the
test compound.
[0026] In a further embodiment, the effect of a test compound on
transcription of a particular lentivirus gene can be determined by
a transfection assay, which uses a reporter gene operatively linked
to at least a portion of the promoter of a lentivirus gene.
[0027] A preferred in vivo assay for identifying a compound which
is useful for treating or preventing a disease or condition
associated with lentivirus infection is comprised of the steps of:
a) administering a test compound to a lentivirus transgenic animal;
and (b) observing at least one phenotype associated with infection
by the lentivirus, wherein a change in phenotype indicates that the
test compound is capable of treating or preventing the disease or
condition. In a preferred embodiment for identifying an effective
vaccine, the transgenic non-human animal is made with an infectious
lentivirus transgene, the compound is a lentivirus antigen or
combination of antigens and the phenotype is an immune response. In
a particularly preferred embodiment for identifying effective HIV
vaccines, the transgenic non-human animal is made with an
infectious HIV transgene, alone or in conjunction with a transgene
encoding a CD4 receptor (e.g. a human CD4 receptor) and/or an HIV
co-receptor transgene (e.g. CCR5 or CXCR4).
[0028] In a further aspect, the invention features methods for
treating subjects infected with a lentivirus or preventing
infection by a lentivirus, comprising administering to the subject
an effective amount of a compound identified according to an assay
of the invention.
[0029] The invention is based at least on the preparation of rats
that are transgenic for HIV proviruses that are deficient in gag
and pol and thus do not produce infectious HIV, and a rat that is
transgenic for human CD4.
[0030] Other aspects of the invention are described below or will
be apparent to those skilled in the art in light of the present
disclosure.
BRIEF DESCRIPTION OF THE FIGURES
[0031] FIG. 1 is a schematic diagram of the HIV-1 proviral DNA used
to prepare HIV-1 transgenic rats as described in the Examples.
[0032] FIGS. 2A and B show the result of indirect
immunofluorescence and flow cytometry analysis of peripheral blood
mononuclear cells (PBMCs) from HIV-transgenic rats, indicating that
the PBMCs express gp120 on their surface.
[0033] FIGS. 3A and B show the results of immunofluorescence and
flow cytometry analyses of PBMCs from a human CD4 transgenic rat,
indicating that the PMBCs express CD4 on their surface.
[0034] FIG. 4A represents a Western blot incubated with anti-HIV-1
Tat antibodies. Lanes 1-8 are as follows: non-transgenic control;
HIV-Tat protein (100 ng; positive control); TgL T cells; TgL B
cells; TgL macrophages; TgH T cells; TgH B cells; and TgH
macrophages. 14.3K and 21.5K mark the location of lysozyme and
trypsin inhibitor proteins from Rainbow.TM. colored protein
molecular weight markers (RPN 756).
[0035] FIG. 4B represents a Western blot incubated with anti-HIV-1
gp120 antibodies. Lanes 1-9 are as follows: non transgenic control;
HIV-1 gp120 protein (30 ng; positive control); TgL macrophages; TgL
T cells; TgL B cells; TgH macrophages; TgH T cells; and TgH B
cells.
[0036] FIG. 5A shows an haematoxylin/eosin (H&E) staining of a
tissue section of a lung from a TgH rat. The arrow indicates an
area of interstitial thickening (original magnification
.times.20).
[0037] FIG. 5B shows an H&E staining of a tissue section of a
mesenteric lymph node from a TgH rat, showing some hemorrhage,
lymphoid depletion and vascular proliferation (original
magnification .times.10).
[0038] FIG. 5C shows skin lesions of a TgH rat, in which gross
lesions on the tail and feet are apparent.
[0039] FIG. 5D shows an H&E staining of a tissue section of
skin from a TgH rat, in which psoriatic skin lesions with
hyperkeratosis and mononuclear cell infiltrate are visible
(original magnification .times.60)
[0040] FIG. 5E shows an H&E staining of a tissue section of a
kidney from a TgH rat, in which focal glomersclerosis and
tubulointerstitial disease are visible (original magnification
.times.60).
[0041] FIG. 5F shows Apotag staining (Oncor, Gaithersburg, Md.) of
blood vessel in the brain from a TgH rat. Arrow indicates vascular
endothelial apoptosis (original magnification .times.60).
[0042] FIG. 5G shows atrocyte staining in a TgH rat with antibody
to GFAP, in which the dark staining areas are astrocytes. The
chromagen used was DAB intensified with cobalt. This reactive
gliosis is a marker for CNS damage (original magnification
.times.40).
[0043] FIG. 5H shows astrocyte staining in a normal control rat, as
described in the legend to FIG. 5G. Here, only limited staining of
the astrocytes is visible, which is staining that is consistent
with normal brain(original magnification .times.40).
[0044] FIG. 5I shows an H&E staining of a tissue section of a
heart from a TgH rat, in which myocardial inflammation with
mononuclear cell infiltration is visible (original magnification
.times.60).
[0045] FIG. 5J shows an H&E staining of a tissue section of a
TgH rat, in which hyperplasia in splenic follicles, and loss of
architecture are visible (original magnification .times.60).
[0046] FIG. 5K shows an H&E staining of a splenic follicle from
a TgL rat, in which hyperplasia in the marginal zone is visible,
and shown by an asterisk (original magnification .times.60).
[0047] FIG. 5L shows an H&E staining of a splenic follicle from
a normal control rat (original magnification .times.60).
[0048] FIG. 5M shows staining of a tissue section of the spleen of
a TgH rat for proliferating cell nuclear antigen (PCNA), in which
dark areas of nuclear staining indicating proliferation are visible
(original magnification .times.10).
[0049] FIG. 5N shows staining of a tissue section of the spleen of
a TgL rat for proliferating cell nuclear antigen (PCNA), in which
dark areas of nuclear staining indicating proliferation are visible
(original magnification .times.20).
[0050] FIG. 6 is a histogram representing the number of apoptotic
cells per high-power field in spleens of HIV transgenic rats TgH
("Heavy") and control rats ("control").
[0051] FIG. 7A is a histogram representing the concentration of
TNF-.alpha. produced by cells of TgH ("HC") and control ("control")
rats 4 or 7 days after stimulation with 10 .mu.g/ml PHA.
[0052] FIG. 7B is a histogram representing the concentration of
IFN-.gamma. produced by cells of a TgH rat ("HC") and a control
("Control") rat 4 and 7 days after stimulation with 10 .mu.g/ml
PHA.
[0053] FIG. 8 is a histogram showing the .sup.3H incorporation
counts at day 5 of a T cell proliferation assays in which
splenocytes from TgH transgenic rats ("HC f" and "HC m") or control
rats ("Crt") were incubated with 10 or 3 .mu.g/ml PHA
DETAILED DESCRIPTION OF THE INVENTION
[0054] General
[0055] The invention is based at least in part on the generation of
HIV transgenic rats, which develop characteristic AIDS symptoms and
that express the HIV envelope protein, gp120, as well as a rat,
which is transgenic for human CD4.
[0056] Definitions
[0057] For convenience, the meaning of certain terms and phrases
employed in the specification, examples and appended claims are
provided below.
[0058] "Antigen" refers to a protein, polypeptide, peptide or other
molecule, which is capable of eliciting an immune response when
administered to a vertebrate.
[0059] "Animal line" refers to a group of animals that are direct
descendants of one founder animal and which bear one or more
transgenes stably integrated into one or more loci in their
germline.
[0060] A "DNA construct" refers to a DNA molecule comprising a
transgene.
[0061] "Founder" generally refers to a first transgenic animal,
which has been obtained from any of a variety of methods, e.g.,
pronuclei injection.
[0062] "Genome" is intended to include the entire DNA complement of
an organism, including the nuclear DNA component, chromosomal or
extrachromosomal DNA, as well as the cytoplasmic domain (e.g.,
mitochondrial DNA). The genome of a eukaryotic organisms, in
contrast to bacterial and viral organisms, is usually arranged into
chromosomes within the cell nucleus.
[0063] "gp-120 is expressed on the surface of a cell" or "a cell
expressing gp-120 on its surface" refers to a cell, which contains
at least one molecule of gp-120 on its surface, preferably at least
about 10, at least about 100, at least about 1000, at least about
10,000 or at least about 100,000 gp-120 molecules on its surface.
Preferred cells expressing gp-120 are those on which gp-120 can be
detected by flow cytometry using an anti-gp 120 antibody. Other
preferred cells expressing gp-120 are cells that have a biological
activity typical of cells having gp-120 on their surface, e.g.,
interaction with CD4 and/or which are capable of syncytium
formation.
[0064] "Heterologous DNA", which is used interchangeably with
"exogenous DNA" refers to DNA that is not naturally present in the
cell.
[0065] The term "HIV" is used interchangeably herein with the terms
"LAV", "LAV-2", "HTLV-III", and "ARV" to refer to human
immunodeficiency virus (HIV). HIV includes both type 1 and type 2
human immunodeficiency viruses and their strains, unless it is used
within the context of a specific embodiment related to type 1 or
type 2 virus. The terms "HIV-1" and "HIV-2" are used to distinguish
the type 1 virus and its strains from the type 2 virus and its
strains. The HIV-1 and HIV-2 genomes, and the DNA sequences of
HIV-1 and HIV-2, and respective strains are further described
herein, as well as in the publication "Human Retrovirus And AIDS
1991", Eds. G. Myer et al., Theoretical Biology and Biophysics, Los
Alamos National Laboratory, Los Almos, N.M., 87545, USA. Nucleotide
sequences of HIV strains can be found in Genbank under the
following Accession Nos: 1) HIV-1: K03455, M19921, K02013, M38431,
M38429, K02007 and M17449; 2) HIV-2: M30502, J04542, M30895,
J04498, M15390, M31113 and L07625 from J. M. Coffin, S. H. Hughes,
and H. E. Varmus, "Retroviruses" Cold Spring Harbor Laboratory
Press, 1997, p 804). In addition to HIV sequences available from
Genbank as described above, HIV sequences can also be found, e.g.,
in the HIV sequence database publically available at
hiv-web.lanl.gov. A map of the HIV-1 genome and transcripts can be
found, e.g., in J. M. Coffin, S. H. Hughes, and H. E. Varmus,
"Retroviruses" Cold Spring Harbor Laboratory Press, 1997, p803).
Set forth in Table 1 is the name and nucleotide location of the
major genes of HIV-1 (from Coffin et al., supra pp 802, 804):
1TABLE 1 name nucleotides comments R 1-96 Repeat is a short
sequence containing the transactivator response region (TAR, i.e.,
Tat Responsive) U5 97-181 PBS 182-199 gag 336-1836 encodes Pr55 Gag
pro 1637-2099 encodes a Pr160 Gag-Pro-Pol precursor pol 2102-4640
pol gene products are synthesized as part of Pr160 vif 4587-5163
encodes p23 Vif protein vpr 5105-5339 encodes p15 Vpr protein tat
5377-5591 encodes p14 Tat protein; binds to the Tat 7925-7968
region of R rev 5516-5591 encodes p19 Rev protein 7925-8197 vpu
5608-5854 encodes p16 Vpu protein env 5771-8339 encodes the gPr160
Env precursor nef 8343-8710 encodes p27 Nef PPT 8615-8630 serves as
principal primer for plus strand synthesis U3 8631-9085 R
9086-9181
[0066] Amino acid sequences of HIV-1 proteins can also be found in
Genbank under the following Accession Nos.(from Coffin et al.,
supra p 804): gag-P04591; pol-P04585; env-p04578; vif-p03401;
vpr-p05926; vpu-p05919; tat-p04608; rev-p04618; and nef-p04601.
HIV-2 has basically the same molecular organization as HIV-1.
However, contrary to HIV-1, HIV-2 contains a gene called vpx, which
encodes a 14 kDa protein of unknown function. Another difference is
that HIV-2 does not contain the vpu gene, which is present in the
HIV-1 genome. Another difference between HIV-1 and HIV-2 is the
presence of a large insertion in the HIV-2 rev gene. There are also
significant differences between HIV-1 and HIV-2 rev in addition to
this insertion.
[0067] In addition, a human T cell line that produces HIV is
available under ATCC Designation No. CRL-8543. A vector containing
the full length HIV-1 genome is available under ATCC Designation
No. 53069. DNA encoding specific HIV genes is also available from
the ATCC, e.g., a clone of human TAR (HIV) RNA binding protein 1 is
available under ATCC Designation No. 107237 and a DNA encoding
env-3 from HIV-1 is available under ATCC Designation No. 53072.
[0068] "Homology" or "identity" or "similarity" refers to sequence
similarity between two peptides or between two nucleic acid
molecules. Homology can be determined by comparing a position in
each sequence, which may be aligned for purposes of comparison.
When a position in the compared sequence is occupied by the same
base or amino acid, then the molecules are identical at that
position. A degree of homology or similarity or identity between
nucleic acid sequences is a function of the number of identical or
matching nucleotides at positions shared by the nucleic acid
sequences. Two DNA sequences are "substantially homologous" or
"substantially similar" when at least about 75% (preferably at
least about 80%, and most preferably at least about 90 or 95%) of
the nucleotides match over the defined length of the DNA sequences.
Sequences that are substantially homologous can be identified by
comparing the sequences using standard software available in
sequence data banks, or in a Southern hybridization experiment
under, for example, stringent conditions as defined for that
particular system. Defining appropriate hybridization conditions is
within the skill of the art. See, e.g., Maniatis et al., supra; DNA
Cloning, Vols. I & II, supra; Nucleic Acid Hybridization,
supra. A degree of identity of amino acid sequences is a function
of the number of identical amino acids at positions shared by the
amino acid sequences. A degree of homology or similarity of amino
acid sequences is a function of the number of amino acids, i.e.
structurally related, at positions shared by the amino acid
sequences. An "unrelated" or "non-homologous" sequence shares less
than 40% identity, though preferably less than 25% identity, with
one of the sequences of the present invention. Two amino acid
sequences are "substantially homologous" or "substantially similar"
when greater than 70% of the amino acids are identical, or
functionally identical. Preferably, the similar or homologous
sequences are identified by alignment using, for example, the GCG
(Genetics Computer Group, Program Manual for the GCG Package,
Version 7, Madison, Wis.) pileup program.
[0069] An "infectious" virus or virus particle refers to a virus
that is capable of replicating and producing new viral particles
when it infects an appropriate cell.
[0070] An "inbred animal line" is intended to refer to animals that
are genetically identical at all endogenous loci.
[0071] The term "isolated" as used herein with respect to nucleic
acids, such as DNA or RNA, refers to molecules separated from other
DNAs, or RNAs, respectively, that are present in the natural source
of the macromolecule. The term isolated as used herein also refers
to a nucleic acid or peptide that is substantially free of cellular
material, viral material, or culture medium when produced by
recombinant DNA techniques, or chemical precursors or other
chemicals when chemically synthesized. Moreover, an "isolated
nucleic acid" is meant to include nucleic acid fragments which are
not naturally occurring as fragments and would not be found in the
natural state. The term "isolated" is also used herein to refer to
polypeptides which are isolated from other cellular proteins and is
meant to encompass both purified and recombinant polypeptides.
[0072] "Lentiviruses" include primate lentiviruses, e.g., human
immunodeficiency virus types 1 and 2 (HIV-1/HIV-2); simian
immunodeficiency virus (SIV) from Chimpanzee (SIVcpz), Sooty
mangabey (SIVsmm), African Green Monkey (SIVagm), Syke's monkey
(SIVsyk), Mandrill (SIVmnd) and Macaque (SIVmac). Lentiviruses also
include feline lentiviruses, e.g., Feline immunodeficiency virus
(FIV); Bovine lentiviruses, e.g., Bovine immunodeficiency virus
(BIV); Ovine lentiviruses, e.g., Maedi/Visna virus (MVV) and
Caprine arthritis encephalitis virus (CAEV); and Equine
lentiviruses, e.g., Equine infectious anemia virus (EIAV). All
lentiviruses express at least two additional regulatory proteins
(Tat, Rev) in addition to Gag, Pol, and Env proteins. Primate
lentiviruses produce other accessory proteins including Nef, Vpr,
Vpu, Vpx, and Vif. Generally, lentiviruses are the causative agents
of a variety of disease, including, in addition to
immunodeficiency, neurological degeneration, and arthritis.
Nucleotide sequences of the various lentiviruses can be found in
Genbank under the following Accession Nos.(from J. M. Coffin, S. H.
Hughes, and H. E. Varmus, "Retroviruses" Cold Spring Harbor
Laboratory Press, 199,7 p 804): 1) HIV-1: K03455, M19921, K02013,
M38431, M38429, K02007 and M17449; 2) HIV-2: M30502, J04542,
M30895, J04498, M15390, M31113 and L07625; 3) SIV:M29975, M30931,
M58410, M66437, L06042, M33262, M19499, M32741, M31345 and L03295;
4) FIV: M25381, M36968 and U11820; 5)BIV: M32690; 6)EIAV: M16575,
M87581 and U01866; 6)Visna: M10608, M51543, L06906, M60609 and
M60610; 7) CAEV: M33677; and 8) Ovine lentivirus M31646 and M34193.
Lentiviral DNA can also be obtained from the American Type Culture
Collection (ATCC). For example, feline immunodeficiency virus is
available under ATCC Designation No. VR-2333 and VR-3112. Equine
infectious anemia virus A is available under ATCC Designation No.
VR-778. Caprine arthritis-encephalitis virus is available under
ATCC Designation No. VR-905. Visna virus is available under ATCC
Designation No. VR-779.
[0073] "Nucleic acid" refers to polynucleotides such as
deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic
acid (RNA). The term should also be understood to include, as
equivalents, analogs of either RNA or DNA made from nucleotide
analogs, and, as applicable to the embodiment being described,
single (sense or antisense) and double-stranded
polynucleotides.
[0074] "Non-infectious" virus or virus particle refers to a virus
that is incapable of producing new viral particles even when it
infects an appropriate cell. A non-infectious human
immunodeficiency virus typically has a deletion in gag and/or pol
and is thereby incapable of replicating and encapsidating the viral
DNA.
[0075] "Phenotype" refers to an observable property of an organism
(in contrast to the genotype, i.e. genetic composition of the
organism).
[0076] The terms "protein", "polypeptide" and "peptide" are used
interchangeably herein when referring to a gene product.
[0077] The term "proviral" DNA refers to a form of a virus that is
integrated into the genetic material of a host cell and by
replicating with it can be transmitted from one cell generation to
the next.
[0078] "Small molecule" as used herein, is meant to refer to a
composition, which has a molecular weight of less than about 5 kD
and most preferably less than about 4 kD. Small molecules can be
nucleic acids, peptides, polypeptides, peptidomimetics,
carbohydrates, lipids or other organic (carbon containing) or
inorganic molecules. Many pharmaceutical companies have extensive
libraries of chemical and/or biological mixtures, often fungal,
bacterial, or algal extracts, which can be screened with any of the
assays of the invention to identify compounds that inhibit viral
gene expression, virus replication, and/or viral production.
[0079] DNA "regulatory elements" include transcriptional and
translational control elements, such as promoters, enhancers,
silencers, terminators, and the like, that provide for translation
or expression of a nucleic acid. In eukaryotic cells,
polyadenylation signals are control sequences.
[0080] The phrase "therapeutically effective amount" as used herein
refers to an amount sufficient to improve by at least about 15
percent, preferably by at least about 50 percent, more preferably
by at least about 90 percent, and most preferably by about 100%
(i.e., cure) a medical condition or symptoms thereof in a subject.
Alternatively the therapeutically effective amount can be an amount
sufficient to reduce by at least about 15 percent, preferably by at
least about 50 percent, more preferably by at least about 90
percent, and most preferably by about 100% the viral load,
expression of a gene, e.g, a viral gene, or viral replication.
[0081] A cell has been "transfected" by exogenous or heterologous
DNA when such DNA has been introduced into the cell. A cell has
been "transformed" by exogenous or heterologous DNA when the
transfected DNA effects a phenotypic change. Preferably, the
transforming DNA should be integrated (covalently linked) into
chromosomal DNA making up the genome of the cell.
[0082] The term "transgene" broadly refers to any nucleic acid that
is introduced into an animal's genome, including but not limited to
genes or DNA having sequences which are perhaps not normally
present in the genome, genes which are present, but not normally
transcribed and translated ("expressed") in a given genome, or any
other gene or DNA which one desires to introduce into the genome.
This may include genes which may normally be present in the
nontransgenic genome but which one desires to have altered in
expression, or which one desires to introduce in an altered or
variant form. A transgene can include one or more transcriptional
regulatory sequences and any other nucleic acid, such as introns,
that may be necessary for optimal expression of a selected nucleic
acid. A preferred transgene of the invention is a viral transgene,
e.g., a lentiviral transgene. A transgene can be as few as a couple
of nucleotides long, but is preferably at least about 50, 100, 150,
200, 250, 300, 350, 400, or 500 nucleotides long or even longer and
can be, e.g., an entire viral genome. A transgene can be coding or
non-coding sequences, or a combination thereof. A transgene usually
comprises a regulatory element that is capable of driving the
expression of one or more transgenes under appropriate conditions.
A "lentiviral transgene" refers to a nucleic acid comprising a
nucleotide sequence encoding at least one lentiviral protein or
biologically active portion thereof. An "HIV transgene" refers to a
nucleic acid comprising a nucleotide sequence encoding at least one
HIV protein or biologically active portion thereof.
[0083] A "transgenic animal" refers to any animal, preferably a
non-human mammal (e.g. mouse, rat, rabbit, squirrel, hamster,
rabbits, guinea pigs, pigs, micro-pigs, prairie, baboons, squirrel
monkeys and chimpanzees, etc), bird or an amphibian, in which one
or more cells contain heterologous nucleic acid introduced by way
of human intervention, such as by transgenic techniques well known
in the art. The nucleic acid is introduced into the cell, directly
or indirectly, by introduction into a precursor of the cell, by way
of deliberate genetic manipulation, such as by microinjection or by
infection with a recombinant virus. The term genetic manipulation
does not include classical cross-breeding, or in vitro
fertilization, but rather is directed to the introduction of a
recombinant DNA molecule. This molecule may be integrated within a
chromosome, or it may be extrachromosomally replicating DNA. In the
typical transgenic animals described herein, the transgene causes
cells to express a viral gene. However, transgenic animals in which
the transgene is silent are also contemplated, as for example, the
FLP or CRE recombinase dependent constructs.
[0084] The term "treating" as used herein is intended to encompass
curing as well as ameliorating at least one symptom of the
condition or disease.
[0085] A "vaccine" refers to a preparation containing at least one
lentiviral antigen, which can be administered to a subject to
produce or artificially increase immunity to a disease, which is
caused by or contributed to by a lentivirus. In addition to the at
least one antigen, the vaccine can optionally comprise a
pharmaceutically acceptable carrier and/or an adjuvant.
[0086] The term "wild-type viral gene or genome" refers to a viral
gene or genome as it is found in nature, i.e., which has not been
manipulated by man. Thus, there may exist several wild-type genomes
for each type of virus.
[0087] Lentiviral Transgenes and Transgenic Animals Produced
Therewith
[0088] The invention provides for transgenic non-human animals
comprising a lentivirus transgene, (e.g., an HIV transgene). The
lentiviral construct can be an infectious virus, which is capable
of replicating and producing viral particles. An example of an
infectious HIV-1 DNA includes the DNA construct referred to as
pNL4-3 (Adachi et al. (1986) J. Virol. 59:284 and Leonard et al.
(1988) Science 242:1665; Genbank Accession No. M19921). Another
infectious HIV-1 proviral construct is pNL4-32 (Strebel et al.
(1987) J. Virol. 328:728 and Leonard et al., supra). Transgenic
non-human animals made with infectious HIV transgenes, alone or in
conjunction with a transgene encoding a CD4 receptor (e.g. the
human CD4 receptor) and/or an HIV co-receptor transgene (e.g. CCR5
or CXCR4) can produce infectious viral particles, which infect host
cells, and therefore are particularly preferred for developing
effective HIV vaccines and therapeutics.
[0089] Non-human transgenic animals, which are noninfectious and
therefore potentially safer for use, can be generated using
transgenes comprised of non-infectious viral DNA, i.e., viral DNA
which does not result in the formation of viral particles upon
infection of a host cell. For example, a non-infectious viral DNA
can have a deletion or other type of mutation in any coding region
or regulatory region sufficient to impair viral nucleic acid
replication, and/or assembly of virions. The deletion can inhibit
production of, or inactivate, one or more of the proteins selected
from the group consisting of a nucleocapsid-core factor (e.g.,
gag), reverse transcriptase, protease, integrase, ribonuclease, and
transcriptional activator (e.g., tat). An example of an HIV-1
provirus that is non-infectious is the pNL4-3:d1443 vector, which
is derived from the infectious pNL4-3 vector by deletion of a 3.1
kb sequence overlapping gag and pol (sequences between the SphI and
BalI sites at bases 14434556), but containing env and the other
accessory genes tat, nef, vif, vpr, and vpu, together with the 5'
and 3' long terminal repeats (LTRs). As described in the following
Examples, pNL4-3:d1443 has been used to produce transgenic rats,
which model human AIDS.
[0090] Other non-infectious HIV DNA can be obtained by deleting
portions of one or both LTRs. For example, the HIV DNA sequence can
be prepared by digesting a plasmid clone containing the DNA
sequence of HIV-1 with a restriction enzyme that cleaves the HIV
proviral DNA sequence at sites proximal to its 5' and 3' ends,
thereby removing essential controlling sequences, to yield a
proviral DNA sequence truncated at both ends, so that the eventual
RNA expression from the cleaved fragment is rendered
non-infectious, but still includes those elements required for the
eventual production of at least some viral proteins. In other
words, the HIV genome is modified to lack the sequences necessary
for reverse transcription, integration and/or transcription. The
extent to which the 5' and 3' ends must be truncated to render the
RNA non-infectious can be determined by standard methods (e.g. by
transforming the fragment so obtained into a genomic equivalent of
HIV-1 and testing the resulting virus for cytopathic activity). As
an example, the Sacd restriction enzyme can be used to cleave the
pBH10 plasmid (B. H. Hahn et al., Nature, 312, 166 (1984)) to yield
an HIV-1 genome deleted of the 5' LTR and/or a portion of its 3'
LTR. Non-infectious HIV proviral DNA deleted 5' and/or 3' are
further described in U.S. Pat. No. 5,574,206 by Jolicoeur.
[0091] Another method for obtaining a non-infectious HIV proviral
DNA sequence involves truncating the HIV genomic DNA fragment from
its 5' end to a point on the untranslated 5' leader sequence
located between about 50 nucleotides downstream from the 5' LTR,
but not including the nucleotide marking the beginning of the
splice donor sequence; and truncating the same HIV DNA fragment
from its 3' end sequence to a point located downstream of the nef
gene, so that the complete encoding sequence of the nef gene is
retained and sequences required for virus replication (i.e. the U5,
R and part of the U3 sequences) are deleted.
[0092] Transgenic animals exhibiting tissue specific expression can
be generated, for example, by inserting a tissue specific
regulatory element, such as an enhancer, into the viral transgene.
For example, one of the LTRs or a portion thereof can be replaced
with another promoter and/or enhancer, e.g., a CMV or a Moloney
murine leukemia virus (MLV) promoter and/or enhancer. For example,
the proviral HIV DNA is pNL4-3, in which the two NF-B binding
motifs of the HIV core enhancer sequences from the Moloney murine
Leukemia Virus (Mo-MuLV) LTR by M13 mutagenesis (deleting
nucleotides -129 to -74, with respect to the HIV mRNA cap site and
replacing them with nucleotides -365 to -40 from the Mo-MuLV). This
construct is further described (as pHm4-3) in Dickie et al. (1996)
AIDS Res. Human Retroviruses 12:177, which also describes
transgenic mice containing this construct.
[0093] An LTR of a proviral genome, e.g, HIV proviral genome, can
also be replaced with a mouse mammary tumor virus (MMTV) LTR, which
is known to be tissue specific toward various epithelial and
hematopoietic tissues, some of which naturally support lentivirus
(and especially HIV) replication. (for an example of such a
construct, see, e.g., U.S. Pat. No. 5,574,206 issued Nov. 12, 1996
to Jolicoeur).
[0094] Alternatively, non-human transgenic animals that only
express HIV transgenes in the brain can be generated using brain
specific promoters (e.g. myelin basic protein (MBP) promoter, the
neurofilament protein (NF-L) promoter, the gonadotropin-releasing
hormone promoter, the vasopressin promoter and the neuron-specific
enolase promoter, see So Forss-Petter et al., Neuron, 5, 187,
(1990). Such animals can provide a useful in vivo model to evaluate
the ability of a potential anti-HIV drug to cross the blood-brain
barrier. Other target cells for which specific promoters can be
used are, for example, macrophages, T cells and B cells. Other
tissue specific promoters are well-known in the art, see e.g. R.
Jaenisch, Science, 240, 1468 (1988).
[0095] Non-human transgenic animals containing an inducible
lentiviral transgene (infectious or noninfectious) can be generated
using inducible regulatory elements (e.g. metallothionein
promoter), which are well-known in the art. Lentiviral gene
expression can then be initiated in these animals by administering
to the animal a compound which induces gene expression (e.g. heavy
metals). Another preferred inducible system comprises a
tetracycline-inducible transcriptional activator (U.S. Pat. No.
5,654,168 issued Aug. 5, 1997 to Bujard and Gossen and U.S. Pat.
No. 5,650,298 issued Jul. 22, 1997 to Bujard et al.).
[0096] Double, triple or multimeric transgenic animals, comprise at
least one other transgene in addition to the lentiviral transgene.
In a preferred embodiment, the animal comprises an HIV transgene
and a transgene encoding human CD4 protein or a portion thereof
sufficient for HIV infection of target cells. In one embodiment,
the CD4 protein comprises a portion sufficient for interaction with
gp120. Preferred portions include the CDR2-like region and the
CDR3-like regions of the first domain of the CD4 molecule (see
e.g., Corbeau et al. (1993) J. Immunol. 150:290). Accordingly, in a
preferred embodiment, a CD4 portion comprises amino acids 81-92,
even more preferably, amino acids 66-92 or 68-130 of human CD4
(Kalyanaraman et al. (1990) J. Immunol. 145: 4072; Ohki et al.
(1993) Vaccine 11:682). In another embodiment, the CD4 protein
comprises a portion sufficient for interaction with gp120 and with
a co-receptor, e.g., CCR5 or CXCR4 (see below). The portion of CD4
that is sufficient for interaction with gp120 and/or a co-receptor
can be identified according to methods known in the art, e.g.,
protein-protein interaction experiments. Examples of methods for
determining the level of interaction between gp120 and CD4
polypeptides are described in, e.g., Lundin et al. (1987) J.
Immunol. Methods 97: 93; Brigham-Burke et al. (1992) Anal. Biochem.
205: 125; and Moore (1990) AIDS 4:297.
[0097] The nucleotide sequence of a cDNA encoding human CD4 can be
found, e.g., in Maddon et al. (1985) Cell 42:93. Mice containing a
CD4 transgene are described, e.g., in Wang et al., (1994) Eur. J.
Immunol., 24: 1553 and in Browning et al. (1997) PNAS 94:14637. A
human CD4 transgenic rabbit has been described, e.g, in PCT
application No. PCT/FR93/00598 (WO 94/00568) by Mehtali et al. The
transgene can be a cDNA containing only coding sequences or
including coding and non-coding sequences. Additionally, it can
also contain intronic sequences. The human CD4 gene can also be a
variant of the wild-type CD4 gene. The sequences of such variant
human CD4 genes can be found in the literature as well as in
GenBank.
[0098] Also within the scope of the invention are animals, in
particular rats, which are transgenic for a human CD4 gene, or at
least a portion thereof, such as a portion described above, and do
not include an HIV transgene. Such animals are described in Example
11, and are useful, in particular, for producing human CD4/HIV
transgenic animals (i.e., animals which contain both a human CD4
transgene and an HIV transgene). Such animals are expected to be
able to become infected by HIV, since it has been shown that a rat
fibroblast line expressing hCD4 is infectable with HIV-1
(Pleskoffet al. (1997) J. Virol. 71: 3259), indicating that the rat
CXCR4 is capable of functioning as a coreceptor in cells expressing
hCD4. Therefore, active HIV-1 infection of a huCD4 transgenic rat
or T cells thereof should be possible. In addition, human CD4
transgenic animals can also be used for the assessment of potential
therapeutic and preventive HIV drugs and vaccines, as well as to
study the pathobiology of HIV-1 infection. These animals are
further useful for identifying drugs for treating or preventing
other diseases, e.g., immune disorders, involving CD4. Since CD4 is
involved in T cell responses, which are implicated in numerous
immunological diseases, e.g., autoimmune diseases, the utility of
such animals is very broad.
[0099] In another preferred embodiment, the animal comprises a
transgene, e.g., an HIV transgene or provirus, and a second
transgene, which expresses a co-receptor (e.g. CCR5 or CXCR4). In a
further preferred embodiment, the animal comprises an HIV transgene
and a transgene (e.g. mutant gene), which is involved in a disease
or condition that is associated with AIDS (e.g. hypertension,
Kaposi's sarcoma, cachexia, etc.). For example, the transgenic
animals of the invention can be crossed with hypertensive rats of
the transgenic rat strain TGR(mREN2)27 harboring the murine Ren-2
gene or transgenic rats comprising a transgene encoding human
angiotensinogen and/or human renin (U.S. Pat. No. 5,731,489). These
transgenic rats develop fulminant hypertension at an early age
despite low levels of renin in plasma and kidney (described in,
e.g., Lee et al. (1996) Am J Physiol 270: E919).
[0100] Where one or more genes encoding a protein are used as
transgenes, it may be desirable to operably link the gene to an
appropriate regulatory element, which will allow expression of the
transgene. Regulatory elements, e.g., promoters, enhancers (e.g.
inducible or constitutive), silencers or polyadenylation signals
are well known in the art. Regulatory sequences can be endogenous
regulatory sequences, i.e., regulatory sequences from the same
animal species as that in which it is introduced as a transgene.
The regulatory sequences can also be the natural regulatory
sequence of the gene that is used as a transgene. Accordingly,
regulatory elements for a CD4 transgene can be the natural CD4
regulatory elements and can include 5' flanking sequence of the CD4
gene comprising promoter and enhancer sequences. Thus, in one
embodiment, a transgenic rat comprises a nucleic acid encoding a
human CD4 protein, or a portion thereof sufficient for binding to
gp120 and allow an HIV to infect a cell expressing such a protein,
under the control of a CD4 promoter, enhancer, and optionally,
silencer, e.g., from human, mouse or rat species.
[0101] Alternatively, a transgene can be placed under the control
of an exogenous regulatory element, i.e., a regulatory element,
which is not the normal regulatory element of the transgene. For
example, a human CD4 transgene can be placed under the control of a
promoter that is functional in specific cell types, e.g., in T
lymphocytes and/or in macrophages and/or monocytes. An exemplary
promoter for use in transgenic rats is the rat CD2 or promoter. An
exemplary promoter allowing expression in T lymphocytes is the Ick
promoter. An exemplary promoter that functions in both T
lymphocytes and in macrophages is the CD4 promoter. Alternatively,
the transcriptional regulatory element can be a viral promoter,
e.g., the MMTV LTR. The transgene also preferably contains a
polyadenylation signal (poly A addition sequence), which can
comprise one or a tandem of two to four of the known poly A
addition signal sequences, such as those derived from the SV40
genome, the casein 3' untranslated region or other 3' untranslated
sequences known in the art. A convenient and readily available
source for the poly A addition signal is the commercially available
pSV2neo vector from which the SV40 poly A addition signal sequence
can be cleaved. Regulatory regions allowing for tissue specific
expression are known in the art, and can be obtained using methods
known in the art. In particular, regulatory regions which are
located 5' of the transcription initiation can be isolated by PCR,
or by screening a genomic library. Preferred 5' regions would be of
sufficient length to contain all of the transcriptional elements
necessary to achieve the desired tissue specific expression. Thus,
preferred regulatory regions comprise at least about 1 kb, 2 kb, 3
kb, 4 kb, 5 kb or 10 kb or 5' region of a gene. The transgene can
be prepared using techniques known in the art; for example see J.
Sambrook et al., "Molecular Cloning: A Laboratory Manual", 2nd ed,
Vols 1 to 3, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., USA, 1989.
[0102] Constructs for use as transgenes can first be tested for
expression in cell lines. Where the transcriptional control
elements in the construct are those from a virus, e.g., HIV, it may
be desirable to use a test cell line of the same type as that which
is naturally infected by the virus. For example, when testing a
construct derived from an HIV provirus, it may be desirable to use
a cell line in which HIV is expressed and is preferably capable of
replicating, e.g., T cell lines. Examples of cell lines in which
HIV is known to replicate include primary human PBMC, isolated
macrophages, isolated CD4+ T cells and cultured human cell lines,
such as HeLa and H9. Expression of the transgene and/or production
of viral particles can be detected as further set forth herein.
[0103] Production of Transgenic Non-Human Animals
[0104] In general, transgenic animal lines can be obtained by
generating transgenic animals having incorporated into their genome
at least one transgene, selecting at least one founder from these
animals and breeding the founder or founders to establish at least
one line of transgenic animals having the selected transgene
incorporated into their genome.
[0105] Animals for obtaining eggs or other nucleated cells (e.g.
embryonic stem cells) for generating transgenic animals can be
obtained from standard commercial sources such as Charles River
Laboratories (Wilmington, Mass.), Taconic (Germantown, N.Y.),
Harlan Sprague Dawley (Indianapolis, Ind.).
[0106] Eggs can be obtained from suitable animals, e.g., by
flushing from the oviduct or using techniques described in U.S.
Pat. No. 5,489,742 issued Feb. 6, 1996 to Hammer and Taurog; U.S.
Pat. No. 5,625,125 issued on Apr. 29, 1997 to Bennett et al.;
Gordon et al., 1980, Proc. Natl. Acad. Sci. USA 77:7380-7384;
Gordon & Ruddle, 1981, Science 214: 1244-1246; U.S. Pat. No.
4,873,191 to T. E. Wagner and P. C. Hoppe; U.S. Pat. No. 5,604,131;
Armstrong, et al. (1988) J. of Reproduction, 39:511 or PCT
application No. PCT/FR93/00598 (WO 94/00568) by Mehtali et al.
Preferably, the female is subjected to hormonal conditions
effective to promote superovulation prior to obtaining the
eggs.
[0107] Many techniques can be used to introduce DNA into an egg or
other nucleated cell, including in vitro fertilization using sperm
as a carrier of exogenous DNA ("sperm-mediated gene transfer",
e.g., Lavitrano et al., 1989, Cell 57: 717-723), microinjection,
gene targeting (Thompson et al., 1989, Cell 56: 313-321),
electroporation (Lo, 1983, Mol. Cell. Biol. 3: 1803-1814),
transfection, or retrovirus mediated gene transfer (Van der Putten
et al., 1985, Proc. Natl. Acad. Sci. USA 82: 6148-6152). For a
review of such techniques, see Gordon (1989), Transgenic Animals,
Intl. Rev. Cytol. 115:171-229.
[0108] Except for sperm-mediated gene transfer, eggs should be
fertilized in conjunction with (before, during or after) other
transgene transfer techniques. A preferred method for fertilizing
eggs is by breeding the female with a fertile male. However, eggs
can also be fertilized by in vitro fertilization techniques.
[0109] Fertilized, transgene containing eggs can than be
transferred to pseudopregnant animals, also termed "foster mother
animals", using suitable techniques. Pseudopregnant animals can be
obtained, for example, by placing 40-80 day old female animals,
which are more than 8 weeks of age, in cages with infertile males,
e.g., vasectomized males. The next morning females are checked for
vaginal plugs. Females who have mated with vasectomized males are
held aside until the time of transfer.
[0110] Recipient females can be synchronized, e.g. using GNRH
agonist (GnRH-a): des-gly10, (D-Ala6)-LH-RH Ethylamide,
SigmaChemical Co.,St. Louis, Mo. Alternatively, a unilateral
pregnancy can be achieved by a brief surgical procedure involving
the "peeling" away of the bursa membrane on the left uterine horn.
Injected embryos can then be transferred to the left uterine horn
via the infundibulum. Potential transgenic founders can typically
be identified immediately at birth from the endogenous litter
mates. For generating transgenic animals from embryonic stem cells,
see e.g. Teratocarcinomas and embryonic stem cells, a practical
approach, ed. E. J. Robertson, (IRL Press 1987) or in Potter et al
Proc. Natl. Acad. Sci. USA 81, 7161 (1984), the teachings of which
are incorporated herein by reference.
[0111] Founders that express the gene can then bred to establish a
transgenic line. Accordingly, founder animals can be bred, inbred,
crossbred or outbred to produce colonies of animals of the present
invention. Animals comprising multiple transgenes can be generated
by crossing different founder animals (e.g. an HIV transgenic
animal and a transgenic animal, which expresses human CD4), as well
as by introducing multiple transgenes into an egg or embryonic cell
as described above. Furthermore, embryos from transgenic animals
can be stored as frozen embryos, which are thawed and implanted
into pseudo-pregnant animals when needed (See e.g. Hirabayashi et
al. (1997) Exp Anim 46: 111 and Anzai (1994) Jikken Dobutsu 43:
247).
[0112] The present invention provides for transgenic animals that
carry the transgene in all their cells, as well as animals that
carry the transgene in some, but not all cells, i.e., mosaic
animals. The transgene can be integrated as a single transgene or
in tandem, e.g., head to head tandems, or head to tail or tail to
tail, or as multiple copies.
[0113] The successful expression of the transgene can be detected
by any of several means well known to those skilled in the art.
Non-limiting examples include Northern blot, in situ hybridization
of mRNA analysis, Western blot analysis, immunohistochemistry, and
FACS analysis of protein expression.
[0114] In particular, the expression of the gag protein (p55), the
gag protein cleavage products p24 and p17, the envelope
glycoprotein (gp 160) and the envelope protein cleavage product gp
120 can be detected using specific probes and/or antibodies. At
least some of these antibodies are commercially available. For
example, monospecific anti-gp120 reagent can be obtained, e.g.,
from Biochorm, Seromed Ref. D7324. Sheep anti-gp120 serum of HIV-1
can also be obtained from the AIDS Research and Reference Program
(catalog no. 192), National Institutes of Health, Bethesda, Md.
Human monoclonal anti-gp120 antibodies are also described in U.S.
Pat. No. 5,695,927 issued Dec. 7, 1997 to Masuho et al.
[0115] Animal tissue can also be analyzed directly, e.g., by
preparing tissue sections. In some embodiments, it is preferable to
fix the tissue, e.g., with paraformaldehyde or formalin. Tissue
sections can be prepared frozen, or can be paraffin embedded.
Slides of animal tissue can be used for immunohistochemistry, in
vitro hybridization or for regular histology, e.g., hematoxylin and
eosin staining.
[0116] Virus can be quantitated by reverse transcriptase (RT)
activity, as is well-known in the art. A change in viral load can
also be determined by quantitative assays for plasma HIV RNA using
quantitative RT-PCR as described, e.g, in Van Gemen et al. (1994)
J. Viro. Methods 49:157; Chen et al. (1992) AIDS 6:533.
Alternatively, viral load can be determined by assays for viral
production from isolated PBMCs. Viral production from PBMCs is
determined by cocultruring PBMCs from the subject with H9 cells and
subsequent measurement of HIV titers using an ELISA assay for p24
antigen levels (Popovic et al. (1984) Science 204:497;
PCTIUS97/11202 (W097/49373) by Gallo et al.). To identify lymphoid
cell types expressing viral RNA, peritoneal inflammatory
macrophages derived from the transgenic animals can be cultured ex
vivo and examined by Northern blot analysis.
[0117] Disease Models and Drug Screening Assays
[0118] The invention further provides methods for identifying
(screening), or for determining the safety and/or efficacy of,
lentivirus therapeutics, i.e. compounds which are useful for
treating and/or preventing the development of diseases or
conditions, which are caused by, or contributed to by lentiviral
infection (e.g. AIDS). In addition the assays are useful for
further improving known anti-viral compounds, e.g, by modifying
their structure to increase their stability and/or activity and/or
toxicity.
[0119] In Vitro Cellular Assays
[0120] Cells from the transgenic animals of the invention (e.g.,
HIV transgenic animals, human CD4 transgenic animals, or human
CD4/HIV transgenic animals which can further be transgenic for an
HIV-co-receptor) can be established in culture and immortalized to
establish cell lines. For example, immortalized cell lines can be
established from the livers of transgenic rats, as described in
Bulera et al. (1997) Hepatology 25: 1192. Cell lines from other
types of cells can be established according to methods known in the
art.
[0121] In one cell-based assay, cells expressing a lentivirus
protein (e.g. receptor) on the outer surface of its cellular
membrane can be incubated in the presence of a test compound alone
or in the presence of a test compound and a lentivirus protein
binding partner (e.g. a receptor ligand) and the interaction
between the test compound and the lentivirus protein or between the
lentivirus binding partner (preferably tagged) and the lentivirus
protein can be detected, e.g., using a microphysiometer (McConnell
et al. (1992) Science 257:1906). An interaction between the
lentivirus protein and either the test compound or the lentivirus
protein binding partner can be detected, (e.g. with a
microphysiometer as a change in the acidification of the medium).
This assay system thus provides a means of identifying molecular
antagonists which, for example, function by interfering with a
lentivirus ligand-receptor interaction, as well as molecular
agonist which, for example, function by activating a lentivirus
protein (e.g. receptor).
[0122] Cell based assays can also be used to identify compounds
which modulate expression of a lentivirus gene, modulate
translation of a lentivirus mRNA, or which modulate the stability
of a lentivirus mRNA or protein. Accordingly, a cell which is
capable of expressing a particular lentivirus protein can be
incubated with a test compound and the amount of the lentivirus
protein produced in the cell medium can be measured and compared to
that produced from a cell which has not been contacted with the
test compound. The specificity of the compound for regulating the
expression of the particular lentivirus gene can be confirmed by
various control analyses, e.g., measuring the expression of one or
more control genes. This type of cellular assay can be particularly
useful for determining the efficacy of antisense molecules or
ribozymes.
[0123] In another embodiment, the effect of a test compound on
transcription of a particular lentivirus gene can be determined by
transfection experiments using a reporter gene, which is
operatively linked to at least a portion of the promoter of a
lentivirus gene. A promoter region of a gene can be isolated, e.g.,
from a genomic library according to methods known in the art. The
reporter gene can be any gene encoding a protein which is readily
quantifiable, e.g, the luciferase or CAT gene. Such reporter genes
are well known in the art.
[0124] In Vivo Assays in Transgenic Animals
[0125] In addition to providing cells for in vitro assays, the
transgenic animals themselves (e.g., HIV transgenic animals, human
CD4 transgenic animals, or human CD4/HIV transgenic animals which
can further be transgenic for an HIV-co-receptor) can be used in in
vivo assays to identify lentiviral therapeutics. For example, the
animals can be used in assays to identify compounds which reduce or
inhibit any phase of the lentiviral life cycle, e.g., expression of
one or more viral genes, activity of one or more viral proteins,
glycosylation of one or more viral proteins, processing of one or
more viral proteins, viral replication, assembly of virions, and/or
budding of infectious virions.
[0126] Other therapeutic compounds which can be identified using
the transgenic animals of the invention include compounds which
prevent or ameliorate pathological conditions seen in HIV infected
individuals, e.g., nephropathy, cardiovascular diseases, central
nervous system disorders, bone marrow dysplasia, endothelium
dysfunction in organs, lymphoid depletion of lymph nodes and
thymus, thymic hypoplasia, psoriasis, moysitis, and vasculitis.
[0127] In an exemplary embodiment, the assay comprises
administering a test compound to a transgenic animal of the
invention and comparing a phenotypic change in the animal relative
to a transgenic animal which has not received the test compound.
For example, where the animal is an HIV transgenic animal, the
phenotypic change can be the amelioration in an AIDS related
complex (ARC), cataracts, inflammatory lesions in the central
nervous system (CNV), a mild kidney sclerotic lesion, or a skin
lesion, such as psoratic dermatitis, hyperkerstotic lesions,
Kaposi's sarcoma, cachexia, or any other macroscopic or microscopic
lesions described herein. The effect of a compound on inhibition of
Kaposi's sarcoma can be determined, as described, e.g., in
PCT/US97/11202 (WO97/49373) by Gallo et al. These and other HIV
related symptoms or phenotypes are further described in Leonard et
al. (1988) Science 242:1665.
[0128] In another embodiment, a CD4 or CD4/lentivirus transgenic
animal is used for identifying and/or testing the efficiency of a
vaccine against the lentivirus. For example, a test compound can be
administered to a CD4 transgenic animal prior to, after, or during
infection with the lentivirus, and the amount of infectious virus,
or the level of a protein or RNA thereof is measured, such that a
reduction in the level of virus or RNA or protein thereof, relative
to an animal to whom no test compound was administered, indicates
that the test compound is efficient as a prophylactic (e.g.,
vaccine) or therapeutic against the lentivirus. In a preferred
embodiment, the lentivirus is HIV and the transgenic animal is a
transgenic rat containing in its genome a human CD4 transgene. In
another embodiment, the transgenic animal is transgenic both for an
HWV provirus and a human CD4 gene. This animal can be used for the
same purpose as a CD4 transgenic animal, but may not require
infection with HIV.
[0129] In yet another embodiment, the phenotypic change is the
number of CD4+ T cells or the ratio of CD4+ T cells versus CD8+ T
cells. In HIV infected humans as well as in HIV transgenic mice,
analysis of lymph nodes indicate that the number of CD4+ T cells
decreases and the number of CD8+ T cells increases. Numbers of CD4+
and CD8+ T cells can be determined, for example, by indirect
immunofluorescence and flow cytometry, as described, e.g., in
Santoro et al., supra.
[0130] Alternatively, a phenotypic change, e.g. a change in the
expression level of an HIV gene can be monitored. The HIV RNA can
be selected from the group consisting of gag mRNA, gag-pro-pol
mRNA, vif mRNA, vpr mRNA, tat mRNA, rev mRNA, vpu/env mRNA, nef
mRNA, and vpx mRNA. The HIV protein can be selected from the group
consisting of Pr55 Gag and fragments thereof (p17 MA, p24 CA, p7
NC, p1, p9, p6, and p2), Pr160 Gag-Pro-Pol, and fragments thereof
(p10 PR, p51 RT, p66 RT, p32 IN), p23 Vif, p15 Vpr, p14 Tat, p19
Rev, p16 Vpu, gPr 160 Env or fragments thereof (gp120 SU and
gp41TM), p27 Nef, and p14 Vpx or unspliced or partially spliced
precursor RNAs thereof. The level of any of these mRNAs or proteins
can be determined in cells from a tissue sample, such as a skin
biopsy, as described in, e.g., PCT/US97/11202 (WO97/49373) by Gallo
et al. Quantitation of HIV mRNA and protein is further described
elsewhere herein and also in, e.g., Dickie et al. (1996) AIDS Res.
Human Retroviruses 12:1103. In a preferred embodiment, the level of
gp120 on the surface of PBMC is determined. This can be done, as
described in the examples, e.g., by immunofluorescence on PBMC
obtained from the animals.
[0131] For example, the proteins expressed in the cells of a
transgenic animal of the invention may include processed gag
proteins resulting from the cleavage of the HIV-1 encoded gag-pol
gene, the cleavage being effected by the HIV-1 encoded protease.
Thus, in one embodiment, the invention provides a method for
evaluating a test compound as a potential HWV-1 protease inhibitor.
In an exemplary embodiment, the method involves: (a) administering
a test compound to the transgenic animal, and (b) examining the
effect of the test compound on the expressed gag proteins in the
animal by monitoring the expression levels of the proteins or RNAs.
The presence of the RNA transcript and the presence or decrease (or
inhibition) of the processed proteins in the cells serves as a
means for evaluating HIV protease inhibitors.
[0132] Likewise, since the presence of the gag and envelope
proteins in the fluid and tissues of the transgenic animal denotes
that the HIV regulatory protein, rev, is expressed, the present
invention provides a method for evaluating a test compound as a
potential inhibitor of rev function. In an exemplary embodiment,
the method involves: (a) administering a test compound to the
transgenic animal, and (b) examining the effect of the test
compound on the expressed gag and envelope proteins and the gag
protein cleavage products in the animal by monitoring the
expression levels thereof.
[0133] A further phenotypic change is the production level or rate
of viral particles in the serum and/or tissue of the animal. This
can be determined, e.g., by determining reverse transcriptase (RT
activity) or viral load as described elsewhere herein as well as in
PCT/US97/11202 (WO97/49373) by Gallo et al., such as by determining
p24 antigen.
[0134] Yet another phenotypic change, which can indicate HIV
infection or AIDS progression is the production of inflammatory
cytokines such as IL-6, IL-8 and TNF; thus, efficacy of a compound
as an anti-HIV therapeutic can be assessed by ELISA tests for the
reduction of serum levels of any or all of these cytokines.
[0135] A vaccine can be tested by administering a test antigen to a
transgenic animal of the invention. The animal can optionally be
boosted with the same or a different antigen. The production of
viral particles or expression of viral proteins is then measured at
various times following the administration of the test vaccine. A
decrease in the amount of viral particles produced or viral
expression will indicate that the test vaccine is efficient in
reducing or inhibiting viral production and/or expression. The
amount of antibody produced by the animal in response to the
vaccine antigen can also be determined according to methods known
in the art and provides a relative indication of the immunogenicity
of the particular antigen.
[0136] Therapeutic and Prophylactic Compounds
[0137] Compounds identified above as being useful for preventing
lentiviral infection and/or treating a lentiviral disease, can be,
e.g. a nucleic acid (e.g DNA, RNA or PNA), protein, peptide,
peptidomimetic, small molecule, or derivative thereof. Preferred
compounds are capable of binding to, and inhibiting transcription,
translation or processing of a lentiviral RNA or protein. Examples
include antisense, ribozyme or triplex nucleic acids, small
molecule ligands, antibody or antibody-like binding fragments).
Alternative compounds are competitive inhibitors of a protein
involved in lentiviral infection, such as a portion of human CD4
sufficient to bind to gp120 and interfere with binding of gp120
proteins on the surface of an infected cell or on the surface of a
viral particle to a human CD4 molecule on the surface of cells.
[0138] Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the Ld.sub.50 (The
Dose Lethal To 50% Of The Population) and the Ed.sub.50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD.sub.50/ED.sub.50. Compounds
which exhibit large therapeutic induces are preferred. While
compounds that exhibit toxic side effects may be used, care should
be taken to design a delivery system that targets such compounds to
the site of affected tissue in order to minimize potential damage
to uninfected cells and, thereby, reduce side effects.
[0139] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED.sub.50 with
little or no toxicity. The dosage may vary within this range
depending upon the dosage form employed and the route of
administration utilized. For any compound used in the method of the
invention, the therapeutically effective dose can be estimated
initially from cell culture assays. A dose may be formulated in
animal models to achieve a circulating plasma concentration range
that includes the IC.sub.50 (i.e., the concentration of the test
compound which achieves a half-maximal inhibition of symptoms) as
determined in cell culture. Such information can be used to more
accurately determine useful doses in humans. Levels in plasma may
be measured, for example, by high performance liquid
chromatography.
[0140] Pharmaceutical compositions for use in accordance with the
present invention may be formulated in conventional manner using
one or more physiologically acceptable carriers or excipients.
Thus, the compounds and their physiologically acceptable salts and
solvates may be formulated for administration by, for example,
injection, inhalation or insufflation (either through the mouth or
the nose) or oral, buccal, parenteral or rectal administration.
[0141] For such therapy, the compounds of the invention can be
formulated for a variety of loads of administration, including
systemic and topical or localized administration. Techniques and
formulations generally may be found in Remmington's Pharmaceutical
Sciences, Meade Publishing Co., Easton, Pa.. For systemic
administration, injection is preferred, including intramuscular,
intravenous, intraperitoneal, and subcutaneous. For injection, the
compounds of the invention can be formulated in liquid solutions,
preferably in physiologically compatible buffers such as Hank's
solution or Ringer's solution. In addition, the compounds may be
formulated in solid form and redissolved or suspended immediately
prior to use. Lyophilized forms are also included.
[0142] For oral administration, the pharmaceutical compositions may
take the form of, for example, tablets or capsules prepared by
conventional means with pharmaceutically acceptable excipients such
as binding agents (e.g., pregelatinised maize starch,
polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers
(e.g., lactose, microcrystalline cellulose or calcium hydrogen
phosphate); lubricants (e.g., magnesium stearate, talc or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g., sodium lauryl sulfate). The tablets may be
coated by methods well known in the art. Liquid preparations for
oral administration may take the form of, for example, solutions,
syrups or suspensions, or they may be presented as a dry product
for constitution with water or other suitable vehicle before use.
Such liquid preparations may be prepared by conventional means with
pharmaceutically acceptable additives such as suspending agents
(e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible
fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous
vehicles (e.g., ationd oil, oily esters, ethyl alcohol or
fractionated vegetable oils); and preservatives (e.g., methyl or
propyl-p-hydroxybenzoates or sorbic acid). The preparations may
also contain buffer salts, flavoring, coloring and sweetening
agents as appropriate.
[0143] Preparations for oral administration may be suitably
formulated to give controlled release of the active compound. For
buccal administration the compositions may take the form of tablets
or lozenges formulated in conventional manner. For administration
by inhalation, the compounds for use according to the present
invention are conveniently delivered in the form of an aerosol
spray presentation from pressurized packs or a nebuliser, with the
use of a suitable propellant, e.g., dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethan- e, carbon dioxide
or other suitable gas. In the case of a pressurized aerosol the
dosage unit may be determined by providing a valve to deliver a
metered amount. Capsules and cartridges of e.g., gelatin for use in
an inhaler or insufflator may be formulated containing a powder mix
of the compound and a suitable powder base such as lactose or
starch.
[0144] The compounds may be formulated for parenteral
administration by injection, e.g., by bolus injection or continuous
infusion. Formulations for injection may be presented in unit
dosage form, e.g., in ampoules or in multi-dose containers, with an
added preservative. The compositions may take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents. Alternatively, the active ingredient may
be in powder form for constitution with a suitable vehicle, e.g.,
sterile pyrogen-free water, before use.
[0145] The compounds may also be formulated in rectal compositions
such as suppositories or retention enemas, e.g., containing
conventional suppository bases such as cocoa butter or other
glycerides.
[0146] In addition to the formulations described previously, the
compounds may also be formulated as a depot preparation. Such long
acting formulations may be administered by implantation (for
example subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compounds may be formulated with
suitable polymeric or hydrophobic materials (for example as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble
salt. Other suitable delivery systems include microspheres which
offer the possibility of local noninvasive delivery of drugs over
an extended period of time. This technology utilizes microspheres
of precapillary size which can be injected via a coronary catheter
into any selected part of the e.g. heart or other organs without
causing inflammation or ischemia. The administered therapeutic is
slowly released from these microspheres and taken up by surrounding
tissue cells (e.g. endothelial cells).
[0147] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration bile
salts and fusidic acid derivatives. in addition, detergents may be
used to facilitate permeation. Transmucosal administration may be
through nasal sprays or using suppositories. For topical
administration, the oligomers of the invention are formulated into
ointments, salves, gels, or creams as generally known in the art. A
wash solution can be used locally to treat an injury or
inflammation to accelerate healing.
[0148] In situations in which the therapeutic is a gene, a gene
delivery system can be introduced into a patient by any of a number
of methods, each of which is familiar in the art. For instance, a
pharmaceutical preparation of the gene delivery system can be
introduced systemically, e.g., by intravenous injection, and
specific transduction of the protein in the target cells occurs
predominantly from specificity of transfection provided by the gene
delivery vehicle, cell-type or tissue-type expression due to the
transcriptional regulatory sequences controlling expression of the
receptor gene, or a combination thereof. In other embodiments,
initial delivery of the recombinant gene is more limited with
introduction into the animal being quite localized. For example,
the gene delivery vehicle can be introduced by catheter (see U.S.
Pat. No. 5,328,470) or by stereotactic injection (e.g., Chen et al.
(1994) PNAS 91: 3054-3057). A therapeutic gene, such as a gene
encoding an antisense RNA or a ribozyme can be delivered in a gene
therapy construct by electroporation using techniques described,
for example, by Dev et al. ((1994) Cancer Treat Rev
20:105-115).
[0149] A gene therapy preparation can consist essentially of a gene
delivery system in an acceptable diluent, or can comprise a slow
release matrix in which the gene delivery vehicle or compound is
imbedded. Alternatively, where the complete gene delivery system
can be produced intact from recombinant cells, e.g., retroviral
vectors, the pharmaceutical preparation can comprise one or more
cells which produce the gene delivery system.
[0150] The compositions may, if desired, be presented in a pack or
dispenser device which may contain one or more unit dosage forms
containing the active ingredient. The pack may for example comprise
metal or plastic foil, such as a blister pack. The pack or
dispenser device may be accompanied by instructions for
administration.
[0151] The present invention is further illustrated by the
following examples which should not be construed as limiting in any
way. The contents of all cited references (including literature
references, issued patents, published and non published patent
applications as cited throughout this application are hereby
expressly incorporated by reference. The practice of the present
invention will employ, unless otherwise indicated, conventional
techniques of cell biology, cell culture, molecular biology,
transgenic biology, microbiology, recombinant DNA, and immunology,
which are within the skill of the art. Such techniques are
explained fully in the literature. See, for example, Molecular
Cloning A Laboratory Manual, 2.sup.nd Ed., ed. by Sambrook, Fritsch
and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA
Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide
Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No.
4,683,195; Nucleic Acid Hybridization(B. D. Hames & S. J.
Higgins eds. 1984); Transcription And Translation (B. D. Hames
& S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I.
Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes
(IRL Press, 1986); B. Perbal, A Practical Guide To Molecular
Cloning (1984); the treatise, Methods In Enzymology (Academic
Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J.
H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor
Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al.
eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer
and Walker, eds., Academic Press, London, 1987); Handbook Of
Experimental Immunology, Volumes I-IV (D. M. Weir and C. C.
Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
EXAMPLES
Example 1
Preparation of an HIV-1 Transgenic Rat
[0152] A transgenic rat containing a proviral HIV-1 DNA, i.e.,
plasmid pNL4-3:d1443, was prepared as follows.
[0153] The HIV-1 proviral DNA in the plasmid pNL4-3:d1443
(represented in FIG. 1) is an artificial recombination of two
strains. The 5' half of pNL4-3 is the viral isolate NY5 and the 3'
half is the viral isolate LAV-1; the recombination splice occurred
at the EcoRi site at nucleotide 5743 base 1 being the most 5'
nucleotide of the LTR). The DNA used for the construction of the
transgenic rat included a deletion of sequence between the BalI and
SphI sites (nucleotides 1443-4551), thereby deleting the gag and
pol genes. The construct included both the 5' and 3' LTRs and open
reading frames for the viral genes, env, tat, rev, nef, vif, vpr,
and vpu. The upstream 5' splice acceptor site is left intact.
Approximately 1 kb of human flanking sequence is present at both
ends. Plasmid pNL4-3 has been described, e.g., in Leonard et al.
(1988) Science 242:1665, illustrating the use of this plasmid for
the preparation of a transgenic mouse.
[0154] Specific pathogen-free inbred Fischer F-344/CrlBR (F344)
rats, and outbred Sprague-Dawley (SD) rats, were purchased from
Charles River Laboratories, Boston, Mass. Animals were maintained
in accordance with institutional guidelines.
[0155] Three week old Fisher 344 (F344) female rats (90-120 g) were
superovulated according to Methods in Molecular Biology Vol. 18,
Transgenesis Techniques, edited by David Murphy and David Carter,
Humana Press pp 253-256. Briefly, the female rats were injected
intraperitoneally with 0.2 IU/g body weight of pregnant mare serum
gonadotropin (PMSG), followed 46-48 hours later with an
intraperitoneal injection of 0.2 IU/g body weight of human
chorionic gonadotropin (HCG). Each female was then placed in a cage
with a stud male. On the morning of the next day, a check was made
for copulatory plugs or vaginal smears were performed to check for
sperm in the lavage. At 12:00 that day, fertilized one-cell eggs
were flushed from the oviducts of females exhibiting either a
vaginal plug or sperm in vaginal lavage fluid. The method for
collecting fertilized eggs was identical to that previously
described for mice (Hogan et al. 1994 and Murphy et al. 1993). Two
types of culture media were used for in vitro manipulations of rat
embryos. M16 was used for maintaining the eggs at 37 .degree. C.
gassed with 5% CO.sub.2. M2 media was used for in vitro
manipulations outside the CO.sub.2 incubator for periods less than
30 minutes. Eggs were collected from the ampulla in M2 media
containing 300 micrograms/ml of hyaluronidase. Following removal of
the cumulus cells, eggs were washed twice in fresh M2 and
transferred to CO.sub.2 equilibrated M16 and incubated at 37
.degree. C. until required for microinjection.
[0156] Pseudopregnant rats were obtained as follows. Female SD
rats, at least 8 weeks of age, were maintained on a 12 hour day and
12 hour night cycle so that they ovulate and mate every 4 days. The
stage of estrous was determined prior to placing them with
vasectomized rats. Sexually mature SD females were anaesthetized
with Methozyflurane, their vagina flushed and the contents dried
and stained with a modified Wright's stain (e.g., Dip-Quick). The
vaginal contents were examined at 40.times. magnification and each
female was staged as to their position in the estrous cycle. SD
females found to be proestrous were placed with vasectomized SD
males, on day 0 by 18:00 hr to generate pseudopregnant
recipients.
[0157] Vasectomized male rats were obtained by a surgical procedure
(Hogan et al., supra). Male rates were anaesthetized with 60 mg/kg
Ketamine and 7.5 mg/kg Xylazine. The abdomen was shaved and cleaned
and the body wall incised and the left and right vas deferens were
cauterized. The body wall was then closed with wound clips and the
rat caged individually in a warm place until recovered.
[0158] Microinjection of Fisher 344 X Sprague Dawley eggs and
transfer to day one pseudopregnant Sprague-Dawley females were
carried out as follows. For injection, the eggs were transferred to
M2 medium. Eggs were sequentially held in place by a blunt pipet
(outside diameter about 100 .mu.m) while the tip of the injector
pipet was inserted through the zona pellucida and vitellus and into
one of the pronuclei. The DNA solution consisting of plasmid
pNL4-3:d1443 at a concentration of 2 ng/ml in the injector pipet
was slowly discharged by using a 100 .mu.l Hamilton syringe
connected to a micrometer. The injector pipet was filled with
silicone oil except for the DNA solution. After injection, the eggs
were transferred to the oviducts of pseudopregnant Sprague-Dawley
female rats. The procedure was identical to that described for mice
(E. Lacy et al., Manipulating the Mouse Embryo, Cold Spring Harbor
Press, N.Y. 1994; and Methods in Molecular Biology Vol. 18,
Transgenesis Techniques, edited by David Murphy and David Carter,
Humana Press). Briefly, the recipient was anaesthetized as
previously described and the oviducts exteriorized by a surgical
incision made at the level of the paralumbar fossa. Approximately
15-30 embryos were transferred per recipient. The body wall was
then closed and the recipient was kept warm until recovery.
[0159] Potential founder transgenic rats were initially identified
by PCR and/or by restriction enzyme digestion and Southern blot
analysis. DNA for PCR or Southern blot analysis was obtained from
2-3 weeks old rat tail tips as per modification of the procedure of
Hogan et al. (E. Lacy et al., Manipulating the Mouse Embryo, Cold
Spring Harbor Press, N.Y. 1994). Approximately, 1 cm long rat tail
tips were excised with a sterile scalpel following anesthesia with
0.02 ml SQ of Lidocaine-HCL. Bleeding was controlled with silver
nitrate. Following tail tip amputations, rats received
Phenylbutazone 50 mg/kg, intraperitoneally as needed for pain. A
Qiagen kit was used to extract DNA from tail tips.
[0160] Identification and quantitation of transgenes was determined
in the founder animals and their progeny by Southern blot analysis
of genomic DNA first amplified by PCR Two primers (SK68 (5' AGC AGC
AGG AAG CAC TAT GG; SEQ ID NO: 4) and SK69 (5' CCA GAC TGT GAG TTG
CAA CAG; SEQ ID NO: 5) were used to specifically amplify a 141 bp
region from HIV-1 env by PCR. Southern blot hybridization with a
.sup.32P labeled 1.2 kb HindIII Nef cDNA fragment was used to
confirm the identification of positive animals. Rats positive for
the transgenic construct are referred to as "TgN(pNL43d14) FO
MBC/IHV TG-1" rats. One female Sprague dawley x Fisher 344/NHsd F1
rat was found to carry the HIV transgene. This founder produced
many hemizygous offspring and brother-sister matings produced
further offspring.
[0161] Southern blot hybridization and PCR analysis indicates that
copies of the proviral HIV genome are inserted in 2 sites in the
genome of the founder rat: one site containing 2-5 copies and a
second site containing 6-25 copies in the genome, respectively.
These two integration sites segregate independently, since the
offsprings from mating of the founder animal with wild-type animals
contain transgenes integrated in either of the two insertion sites.
Thus, among the offspring are rats containing 2-5 copies of the
transgene and rats containing 6-25 copies of the transgene. It is
likely that the transgene is inserted on two different chromosomes.
It is believed that the number of copies of the transgene
correlates with the degree of certain characteristics of the
phenotype of the animal, in particular with the degree of cataracts
(light versus heavy cataracts). The transgenes are inserted at two
separate integration sites which segregate independently. A rat
having 2-5 copies of the transgene has light cataracts while a rat
having 6-25 copies of the transgene has dark cataracts.
Example 2
Phenotype of the HIV Transgenic Rat
[0162] The female founder rat, TG-1, has cataracts in both eyes and
a small red circular lesion at the base of the tail. TG-1 was mated
with a normal Sprague Dawley male to produce the F.sub.1
generation. The F.sub.1 offsprings had cataracts that varied from a
high degree of opacity to a faint one. The cataracts were supplied
with a large number of blood vessels (highly vascular or
angiogenic). Transgenic animals had bilateral cataracts at birth.
Two phenotypes could be distinguished: one phenotype which
consisted of heavy, opaque cataracts ("TgH" rats), and the other
which consisted of light, milder opaque cataracts ("TgL" rats). The
heavy cataracts were found in animals having the transgene
integrated as 6-25 copies, whereas the lighter cataracts were found
in animals having the transgene integrated as 2-5 copies. Thus, the
severity of cataracts in the HIV transgenic rats correlates with
the number of copies of the transgene.
[0163] In addition, at the time of weaning, most animals developed
a focal skin lesion at the base of the tail. Of the 10 in the first
litter three had red lesions at the base of the tail, all were
females. Subsequent offsprings have also demonstrated that a few
males have the red lesion. Mating of this phenotype produce
offsprings with more severe skin lesions that cover the length of
the tail and the offsprings were smaller, especially the females. A
pregnancy factor during early pregnancy (first trimester) causes
the lesion to disappear and return later in partituation. The
severe skin lesion phenotype were smaller, had a larger amount of
proteins in urine, increased BUN and a higher alkaline urine. Skin
lesions were that of psoratic dermatitis and hyperkerastotic
lesions, mild to severe kidney sclerotic lesions, and inflammatory
lesions in the CNS.
[0164] The animals also develop a wasting syndrome, respiratory
problems, and in some a mild to severe neurological disease.
Histological examination also revealed mild to severe dermatitis,
conjunctivitis, atrophy and fibrosis of the thymus, focal global
glomero-sclerosis and microcystic dibtion, cardiomyopathy and
myocarditis, bone marrow dysplasia, vasculitis, mesenteric
lymphanditis, vascular inflammatory disease of the brain, skeletal
muscle degeneration, and pneumonitis. Calcium vasculopathy has been
demonstrated in the brain, spinal cord, blood vessels and kidneys.
Apoptosis of endothelium cells in the brain, pituitary,
conjunctivia and kidney was a common finding in the animals.
[0165] Interestingly, it seems that the transgenic rats having the
integration site with 6-25 copies of the transgene have the
above-described pathological conditions, whereas the rats having
the integration site with 2-5 copies have solely cataracts and do
not show any of the other pathological conditions described above
(or at least these conditions are less apparent than in rats having
6-25 copies of the transgene). Thus, these two types of HIV
transgenic rats may be used as two different animal models. In
addition, the level of serum and membrane bound gp120 in the
animals may correlate with certain pathological conditions and/or
with the level of apoptosis in CD4 cells. In particular, it is
believed that higher serum gp 120 may be responsible for a higher
level of apoptosis of CD4 cells.
[0166] Thus, the HIV transgenic rat of the invention displays many
of the pathology seen in humans with HIV, including retarded
growth, CNS disturbances, mild to severe skin lesions, kidney
problems, encephalitis, cardiovascular abnormalities, bone marrow
displasia, endothelium cell dysfunction in major organs, lymphoid
depletion of lymph nodes and thymus, thymic hypoplasia, psoriasis,
skeletal muscle myositis, and vasculitis. Accordingly, the
transgenic animals of the invention constitute an ideal model for
studying HIV infection and for developing therapeutics for
preventing or treating HIV infections and associated
conditions.
[0167] Since the transgenic rat has a similar phenotype to that of
a transgenic mouse containing the pNL4-3 proviral DNA, the
phenotype of the transgenic rat is most likely not due to an
insertional inactivation event.
Example 3
Expression Pattern of HIV in HIV Transgenic Rats
[0168] Expression of HIV genes in the HIV transgenic rats obtained
as described in Example 1 was determined by RT-PCR as follows.
[0169] Rat tissues (i.e. eye, skin, muscle, brain, bone, heart,
adrenal glands, kidney, large intestine, liver, lung, pancreas,
small intestine, spleen, stomach, testicle, tongue, and thymus)
were necropsied and snap-frozen in liquid nitrogen. The tissues
were stored at -84.degree. C. until processing. The tissues were
homogenized in approximately 1 ml of Trizol (life Technologies)
using a PowerGen Homogenizer (Fisher Scientific). After
homogenation the samples were incubated at room temperature for 5
minutes to permit the complete dissociation of nucleoprotein
complexes. 0.2 ml of chloroform was added to the samples before
shaking for 2-3 minutes by hand. The milky pink samples were then
centrifuged at 12,000 g for 15 minutes at 4.degree. C. The mixture
separated into a lower, red, phenol-chloroform phase, an
interphase, and a colorless upper aqueous phase, which contains the
RNA. The aqueous phase was transferred to a fresh tube, and mixed
with 0.5 ml isopropyl alcohol. The samples were incubated for 10
minutes at room temperature then centrifuged at 12,000 g for 10
minutes at 4.degree. C. The RNA precipitate formed a pellet on the
side and bottom of the tube. The supernatant was removed from the
pellet. The pellet was then washed once with about 1 ml of 75%
ethanol. The sample was then vortexed and centrifuged at 7,500 g
for 5 minutes at 4.degree. C. The supernate was once again removed,
and the pellet was allowed to air-dry for up to 30 minutes. DEPC
water was then added to redissolve the pellet. To assist in
dissolving the pellet, the samples were incubated for 10 minutes
(sometimes longer) at 60.degree. C. The samples were then stored at
-80.degree. C.
[0170] cDNA was prepared from the RNA as follows. 20 .mu.l of the
RNA sample was incubated with 20 U of DNAse I (10 Units/.mu.l) for
1 hour at 37.degree. C. The sample was then phenol extracted
(Trizol method above) to remove the DNA and DNAse protein from the
RNA. The RNA was precipitated with ethanol and sodium acetate
overnight. The pellet was washed, and dissolved as described above.
2 .mu.l of the sample was diluted with 200 .mu.l of deionized
water. The sample was then quantitated to determine the amount of
RNA present. 17 .mu.l was made to contain 2 .mu.g, of Dnased RNA.
On ice, a cocktail of 1 .mu.l Random Heximers 100 M, 6 .mu.l
5.times. Reverse Transcriptase Buffer, 3 .mu.l DTT 0.1M, 1.5 .mu.l
DNTP 10 mM, 0.5 .mu.l RNAse inhibitor, and 1 .mu.l Reverse
Transcriptase (200u) Moloney Murine Leukemia Virus Reverse
Transcriptase was added to the Dnased RNA. For samples reversed
transcribed with the Art7 primer, 1.6 .mu.l Art7 and 5.4 .mu.l
5.times.RT buffer was used. The mixture was incubated for 1 hour at
37.degree. C. The samples were then heated at 95.degree. C. for 5
minutes to kill the enzyme. The samples were immediately put on
ice.
[0171] PCR was performed as follows. 5 .mu.l of cDNA was added to
40 .mu.l of PCR SuperMix (Gibco). The PCR SuperMix contains 22 mM
Tris-HCl (pH 8.4), 55 mM KCl, 1.65 mM MgCl.sub.2, 220 .mu.M dGTP,
220 .mu.M dATP, 220 .mu.M dTTP, 220 .mu.M dCTP, 22 U recombinant
Taq DNA Polymerase/ml, stabilizers. 5 l of 5', 3' primers (20
.mu.M) were added to the reaction mixture. The samples were first
denatured for 3 minutes at 95.degree. C. The parameters for PCR
amplification were as follows: 35 cycles, each with denaturation at
95.degree. C. for 1 minute, annealing at 60.degree. C. for 2
minutes, and extension at 72.degree. C. for 2 minutes. The final
cycle was followed by a 5 minute extension at 72.degree. C. The
samples were then held a 4.degree. C.
[0172] The sequences of the primers and probes used for cDNA
synthesis and detection as well as the expected products are as
described in Bruggeman et al. (1994) Virology 202:940. Since the
mRNA encoding all HIV-1 proteins are processed from the same
precursor RNA with alternative splicing and all HIV-1 mRNAs have
identical 5' exons, the 5' sense primer used for all cDNA synthesis
was US (TAG TAG CAT GCT CTC TCG ACG CAG GAC TCG GCT TGC; SEQ ID NO:
1). The primer pair US and ART7 (ATG ATC TGC AGT TCT ATT CCT TCG
GGC CTG TCG; SEQ ID NO: 3) was used to amplify the tat, rev, and
nef genes. Probing of the amplified products with S1 identifies a
402 bp tat fragment, while probing with S2 identifies 402 bp-tat
and 219/225 bp-rev fragments and probing with S3 identifies 402
bp-tat, 219/225 bp-rev and 203 bp-nef fragments. The primer ART5 is
downstream from the slice acceptor for vif and the primer ART2 is
downstream from the initiation codon of Env. The primer pair
ART5/US amplifies vif mRNA and generates a 338 bp fragment when
probed with S4. Primers ART2 (ACC TCC TGC AGC ACA GGT ACC CCC ATA
ATA GAC TGT G; SEQ ID NO: 2) and US was used to amplify env mRNA
and generated a 446 and 665 bp product when probed with the S3
probe. The 5' and 3' primers for G3PDH and SK68 (5' AGC AGC AGG AAG
CAC TAT GG; SEQ ID NO: 4) and SK69 (5' CCA GAC TGT GAG TTG CAA CAG;
SEQ ID NO: 5) for Env were used to amplify regions of cDNA
generated from random hexamers.
[0173] Following amplification, the reaction mixtures were
subjected to electrophoresis, the nucleic acids were transferred
onto a blot and the blot was hybridized with the following
probes:
2 S1 GAG CCA GTA GAT CCT AGA CTA GAG C; (SEQ ID NO: 6) S2 CTT AGG
CAT CTC CTA TGG CAG GAA; (SEQ ID NO: 7) S3 ACC TCG CAT GCG AAG AAG
CGG AGA CAG CGA CGA AG; (SEQ ID NO: 8) and ENV TGA CGC TGA CGG TAC
AGG CC. (SEQ ID NO: 9)
[0174] The results of tissue expression analysis indicate the
presence of transcripts of about 7 kb (full length gag-pol mRNA), 4
kb (singly spliced env. mRNA) and 2 kb (multiply spliced tat, rev,
and nef mRNA) in numerous tissues including the eyes, skin, and
muscle and moderate expression in the brain and heart, and light
expression in bone and bladder. Some expression was also found in
liver, thymus, kidney, and spleen. No detectable levels of Env mRNA
were found in the adrenal glands, large intestine, lung, pancreas,
small intestine, stomach, testicle, and tongue.
[0175] The level of expression of HIV transcripts in auxiliary and
mesenteric lymph nodes, thymus, livers, kidneys and spleens was
compared in TgH and TgL rats. Total RNA was isolated from various
tissues as previously described (Puissant et al. (1990)
Biotechniques 8: 148) using RNAzolTmB (Tel-Test, Inc., Friendswood,
Tex.). The RNA was separated on a 1% agarose/formaldehyde gel in
1.times.MOPS buffer, then transferred to a Nytran.RTM.SuPerCharge
nylon membrane (Schleicher & Schuell (S&S), Keene, N.H.)
with 10.times.SSC using a TurboBlotter.TM. (S&S). The RNA was
cross-linked to the filter by UV light in a GS Gene Linker.TM.
(BioRad, Hercules, CA). HIV-specific transcripts (7 kb, 4 kb and 2
kb) were detected by hybridization in Ultrahyb.TM. Solution
(Ambion, Inc., Austin, Tex.) with an [.alpha.-.sup.32P]-labeled 1.3
kbp (BglII/BglII) fragment from pHXB2, containing coding regions
for gp41 and nef The relative amounts of the HIV transcripts were
quantified with a Storm 840 Phosphorimager (Molecular Dynamics,
Sunnyvale, Calif.) and normalized for hybridization to 18S rRNA
using an appropriate probe.
[0176] The results of the Northern blot analysis show the three
forms of viral transcripts, representing full length 7.4 kb mRNA,
4.0 kb singly spliced Env mRNA and multiply spliced 2 kb mRNA
transcripts for Nef, Tat and Rev. When these signals were
normalized for the signal from 18S ribosomal RNA, there was little
consistent difference in viral expression between TgH and TgL
tissues. Expression levels were generally highest in auxiliary
lymph nodes, spleen, kidney and thymus. Mesenteric lymph nodes of
the TgH rats gave relatively low levels of expression; this may be
correlated with the moderate to severe lymphocyte depletion
observed in these animals (see Example 7).
[0177] Thus, the tissue distribution of HIV transcripts in the HIV
transgenic rats is similar to that in humans. Expression is similar
in TgH and TgL rats. In addition, the presence of high levels of
Env gp120 and unspliced and singly spliced viral RNA in the Tg rats
suggests that Rev is functional in the HIV transgenic rat.
Example 4
Gp120 is Present in the Serum and on the Surface of PBMCs in HIV
Transgenic Rats
[0178] Expression of the envelope protein in serum and PBMC was
assayed by ELISA capture and flow cytometry, respectively. The
ELISA assay indicated that two of the two hemizygous transgenic
animals TgL contained gp120 in their sera at levels of
approximately 145 .mu.g/ml. Interestingly, rats having 2-5 copies
of the transgene (TgL) have higher levels of serum gp120 than do
rats having 6-25 copies of the transgene (TgH). Indeed, the average
serum concentration of gp120 in TgL rats in three rats was about 9
ng/ml.
[0179] Multicolor flow cytometry of PBMCs was performed on a
FACSCalibur (Becton-Dickinson Mountain View, Calif.) as previously
described (Taurog and El-Zaatari (1988) J Clin Invest 82, 987-92;
Taurog et al. (1988) J Clin Invest 82, 987-92). Briefly,
Ficoll-Hypaque purified peripheral blood mononuclear cells were
incubated with saturating concentrations of F105 human anti-env
antibody (available from the AIDS Repository), washed, then
incubated with anti-human-FITC secondary antibody (available from
Pharmigen Commercial). The staining was done in the presence of 1%
human AB serum (available from Sigma Commercial). After washing,
the cells were fixed in 1% paraformaldehyde before analysis on a
FACScan flow cytometer (Becton Dickinson, Mountain View, Calif.).
Viable lymphocytes were selected for analysis by gating of forward
and 90 light scatter.
[0180] The FACS analysis, which is shown in FIG. 2, indicates that
gp120 was readily detectable on the surface of the entire PBMC
population. Thus, the data indicate that, contrary to mice
transgenic for pNL4-3:d1443, the gp120 env protein is expressed on
the surface of PBMCs of HIV transgenic rats and also shed into
their serum.
Example 5
Serum Anti-Nef and Anti-Gp120 IgG are Present in HIV Transgenic
Rats
[0181] Sera from HIV-1 transgenic rats were assayed by ELISA for
the presence of antibodies to two viral transgene products, Nef and
gp120. The ELISA assay was basically as described by Moore et al.
(1994) J. Virol. 68: 5142. A capture ELISA on twenty transgenic
rats was done in duplicate at serum dilutions of 1:30 and 1:100
along with four negative controls. Positive antibody titers are
absorbance values greater than the mean of the negative controls
plus three standard deviations.
[0182] The results indicate, that, out of the ten light cataract
transgenic rats, two had positive titers at 1:30 serum dilution for
IgG antibodies to Nef alone; three had positive titers at 1:30
serum dilution for IgG antibodies to both Nef and gp120; and none
had positive titers for IgG antibodies to gp120 alone. Out of the
ten heavy cataract transgenic rats, one had positive titers at 1:30
serum dilution for IgG antibodies to Nef alone; three had positive
titers at 1:30 serum dilution for IgG antibodies to both Nef and
gp120; and none had positive titers for IgG antibodies to gp 120
alone.
[0183] These data indicate that nearly half of both groups of
transgenic rats mounted an antibody response to the transgene
products. This apparent lack of immune tolerance suggests that
HIV-1 LTR-driven expression only occurs after maturation of the
immune system, and that transgene expression is relatively
persistent.
Example 6
HIV Transgenic Rats Express Viral Proteins in Macrophages B Cells
and T Cells
[0184] Formalin-fixed paraffin-embedded five-micron sections of
spleen from TgH and TgL rats were analyzed by immunohistochemistry
for expression of HIV-1 gp120, Nef and Tat.
[0185] Tissues from Fisher 344/NHsd control rats, Sprague Dawley
control rats, and TgH and TgL rats were fixed in 10% neutral
buffered formalin (PBS pH 7.2) and embedded in paraffin. Five .mu.m
tissue sections were used for hematoxylin and eosin (H&E)
staining, gp120, Tat and Nef immunohistochemistry. A modified
avidin/biotin method was used for immunohistochemical localization
of HIV gene products. Paraffin sections were collected,
deparaffinized with xylene and hydrated using graded alcohols.
Sections were exposed to antigen unmasking solution (Vector
Laboratories Inc., Burlingame, Calif.) according to the
manufacturer's directions. Endogenous peroxidase was inhibited by
incubation in 3% H.sub.2O.sub.2 for 20 minutes. This was followed
by treatment with avidin/biotin blocking solution (Vector
Laboratories) and non-immune sera appropriate for blocking the
secondary antibody, at a 1:5 dilution. Blocking sera, including
normal goat, horse, and rabbit, were from Vector Laboratories.
Primary antibodies included HIV-1 gp120 Goat Antisera (13-202-000)
(Advanced Biotechnologies), used at 1:50 and 1:100 dilutions, HIV-1
gp120 Rabbit antisera (13-204-000) (Advanced Biotechnologies), used
at a 1:100 dilution, mouse anti-HIV-1 gp120 monoclonal antibody
(NEA 9301) (NEN), used at a 1:150 dilution, mouse anti-HIV-1 Tat
monoclonal antibody (13-162-100) (Advanced Biotechnologies), used
at 1:50 and 1:100 dilutions, and mouse anti-HIV-1 Nef (13-152-1000)
(Advanced Biotechnologies), used at 1:50 and 1:100 dilutions.
Incubations were overnight at 5.degree. C. Biotinylated secondary
antibodies were incubated for 2 hrs at room temperature at
dilutions of 1:200-500, and included anti-mouse IgG (rat-absorbed),
anti-rabbit IgG, and anti-goat IgG (Vector Laboratories). Labeling
was with Vecta Stain Elite ABC (Vector Laboratories), used
according to the manufacturer's instructions, followed by addition
of DAB peroxidase (Sigma, St Louis, Mo.) or the AEC substrate
system (DAKO Corp., Carpinteria, Calif.) to visualize the
immunolabel. A peroxidase-linked antibody to proliferating cell
nuclear antigen (PCNA), a marker for cell proliferation, was used
to identify recently dividing splenocytes (07032) (DAKO Corp.,
Carpinteria, Calif.). Staining for glial fibrillary acid protein
(GFAP) was performed as previously reported (Wiley, C. A. J.
Neuropathol. Exp. Neurol. 45, 127-139 (1986)) and using anti GFAP
antibody (U7038) (DAKO Corp., Carpinteria, Calif.).
[0186] The results show that, in both the TgL and TgH rats, gp120,
Nef and Tat viral proteins are locally expressed in cells within
the red and white pulp of the spleen, and that the apparent
expression of viral proteins is similar in the two groups of
animals despite differences in clinical and microscopic
pathology.
[0187] The level of gp120 and Tat was investigated in macrophages,
B cells and T cells by Western blots as follows. Fresh splenocytes
from TgH and TgL rats were purified on Histopaque.RTM.-1083 (SIGMA,
St. Louis, Mo) and stained with primary monoclonal antibodies by
standard procedure (Current Protocols in Immunology. Joh Wiley and
Sons, New York (1994)). Generally, cells were counted and divided
into three aliquots, each containing approximately
1.5.times.10.sup.7 cells. Each aliquot was labeled for 45 minutes
at room temperature with a 1:100 dilution of anti-rat monoclonal
antibody EDI (MCA341R) (Serotec Inc., Raleigh, N.C.), CD45RA
(MCA340R) (Serotec) or CD3 (22011D) (PharMingen, San Diego,
Calif.), which identify rat macrophages, B-cells and T-cells,
respectively. Following the primary labeling, cells were
counterstained with 20 .mu.l per 10.sup.7 cells of MACS rat
anti-mouse IgG1 (471-02) or goat anti-mouse IgG (484-02) (Miltenyi
Biotec, Auburn, Calif.) for 15 minutes at 6-12.degree. C. The
magnetically labeled cell suspensions were separated by positive
selection on MS+ separation columns placed in a VarioMACS magnet
(Miltenyi Biotec, Auburn, Calif.) using the manufacturer's
recommendations.
[0188] Total protein was extracted from splenocytes enriched for
macrophages, B-cell or T-cells in RIPA buffer (PBS, 1% NP40, 0.5%
sodium deoxycholate, and 0.1% SDS) containing PMSF, aprotinin, and
sodium orthovanadate. Seven .mu.g of protein from each sample was
fractionated on a 4-12% NuPage gel in Tris-MES-SDS buffer (Novex,
San Diego, Calif.), transferred to an Optitran membrane (S&S),
and labeled with a 1:100 dilution of mouse anti-HIV-1 gp120
monoclonal antibody (NEA 9301) (NEN Life Science Products Inc.,
Boston, Mass.) or mouse anti-HIV-1 Tat monoclonal antibody
(13-162-100) (Advanced Biotechnologies, Columbia, Md.) followed by
exposure to a 1:2000 dilution of peroxidase-conjugated goat
anti-mouse IgG (Kirkegaard and Perry Laboratories, Gaithersburg,
Md.). Proteins were visualized using the ECL Plus Western blotting
system (Amersham, Arlington Heights, Ill.).
[0189] The results, which are shown in FIG. 4, indicate that both
Tat and gp120 were present in cellular lysates from all three cell
types. Both groups of animals were also antigenic. Thus, unlike
transgenic mice with the same proviral transgene, TgL and TgH rats
express viral proteins in macrophages, B cells and T cells. In
addition, the relatively high levels of viral gene expression
observed in lymphoid tissues suggest that Tat is functional in the
Tg rats.
Example 7
Pathology of HIV Transgenic Rats
[0190] As described above, in addition to heavy cataracts, TgH rats
developed many of the clinical manifestations of AIDS by five to
nine months of age. These included a wasting syndrome, neurologic
abnormalities and respiratory difficulty. Generally, the neurologic
abnormalities were characterized by a circling behavior and hind
limb paralysis. Ulcerative skin lesions were also often present. In
contrast, TgL rats presented with less severe pathology. A summary
of the pathology of the HIV-1 transgenic rats is set forth in Table
2.
3TABLE 2 Disease associated pathology in the HIV-1 transgenic rats
Clinical Pathology in Transgenic Rats TgH Breathing
Gross/Microscopic Pathology TgL Difficulty Wasting Neuro. Gross
Pitted or small kidney 0/2 2/2 5/5 2/2 Enlarged lymph node 0/2 0/2
3/5 1/2 Enlarged heart 0/2 1/2 3/5 1/2 Enlarged spleen 0/2 2/2 2/5
1/2 Microscopic Spleen (follicular hyperplasia) 1/2 2/2 5/5 2/2
Lung (interstitial pneumonia) 1/2 2/2 3/5 2/2 Lymph nodes
(lymphocyte 0/2 2/2 2/5 1/2 depletion) Kidney (glomerulosclerosis)
0/2 2/2 4/5 2/2 Heart (myocardial degeneration) 1/2 1/2 2/5 1/2 and
or smooth muscle hypertrophy) Brain (neuronal loss and gliosis) 0/2
1/2 0/5 2/2
[0191] The following analyses were undertaken to further analyze
the transgenic rats. Five-micron paraffin-embedded H&E stained
tissue sections, prepared as described in the previous example,
from TgH and TgL rats were examined by light microscopy.
[0192] Generally, the severity of the histopathology of tissues
from TgH rats was similar regardless of their clinical
presentation. TgL rats had predominantly a milder or normal
histopathology. Microscopically, H&E stained tissue sections of
the lung from TgH and TgL rats showed evidence of a mild
interstitial pneumonia characterized by mild to moderate
interstitial fibrosis and mononuclear cell infiltration (FIG. 5A).
The mesenteric lymph nodes in TgH animals were generally enlarged
and many histological sections were characterized by lymphoid
depletion and fibrosis (FIG. 5B). Cataract formation ranged in
severity from light to heavy. Some transgenic animals presented
with highly angiogenic corneas. Their lenses had marked
vacuolization, liquefaction and fragmentation. Many of the TgH rats
had focal to extensive ulcerative skin lesions (FIGS. 5C and 5D).
Histologically the lesions were hyperkerototic, with elongation of
the rete ridges. The kidneys from clinically ill TgH rats were
diffusely pale and enlarged (approximately twice controls, by
weight), and the capsular surface was pitted, similar to what is
seen in patients with HIV-1 associated nephropathy (HIVAN). H&E
stained kidney sections showed a spectrum of renal disease that
varied from mild to severe. Some TgL rats showed only mild
degenerative changes in the proximal convoluted tubules, without
significant glomerular changes. In contrast, the majority of TgH
rats as exemplified by FIG. 5E showed multiple severe renal
lesions, which were consistent with HIVAN in humans. Essentially
all glomeruli in TgH rats contained increased PAS-positive
material, with either segmental or global sclerosis. Some glomeruli
showed mesangial hypercellularity and enlargement of visceral
epithelial cells. Silver staining confirmed that the PAS-positive
tissue within the glomeruli was composed of matrix material. The
renal tubules showed microcystic tubular and tubulointerstitial
pathology characterized by tubular degeneration, interstitial
fibrosis and mononuclear cell infiltration. Many kidneys showed
evidence of moderate to diffuse nephrocalcinosis.
[0193] The brains of the TgH with or without clinical neurologic
signs appeared unremarkable by gross examination. However, a number
of pathologic changes could be identified in H&E stained
sections from rats with clinical signs. Capillaries and endothelial
cells presented with atypical changes, such as microscopic
hemorrhages and endothelial cell apoptosis, in a multifocal
distribution (FIG. 5F). Foci of gliosis together with neuronal cell
death were noted, particularly in the animals with clinically
observable signs (FIG. 5G). For comparison, FIG. 5H shows a normal
section of the brain. While these changes seemed to be distributed
in a random fashion, when they occurred with increased severity in
focal areas of the brain, corresponding neurological deficits were
noted. For example, animals with motor problems presented with
greater severity of changes in the caudate putamen and substantia
nigra.
[0194] The heart of both TgH and TgL rats generally appeared round
and pale in color (not shown). H&E staining in some hearts from
TgH rats showed evidence of endocarditis and myocardial
inflammation characterized by necrosis, mononuclear cell
infiltrates and multiple vascular abnormalities (FIG. 5I). The
spleen from TgH rats was generally enlarged (up to twice the size
of controls). In contrast, spleens from TgL generally appeared
normal in size. H&E staining of spleen tissue sections from TgH
rats showed that the general histological architecture of the
splenic follicles are disrupted in TgH rats (FIG. 5J) and replaced
by a moderate to severe follicular hyperplasia, while in TgL the
spleen was characterized by a mild follicular hyperplasia with
expansion of the marginal zone (FIG. 5K). For comparison, (FIG. 5L)
shows a normal spleen.
[0195] Splenocytes from TgH showed evidence of focal areas of
proliferation as evidenced by staining for proliferating cell
nuclear antigen (PCNA), (FIG. 5M). The amount and distribution of
PCNA staining was more extensive in spleen sections from non-Tg
controls; however, the extent and distribution of PCNA staining in
spleens from non-Tg controls and TgL rats was identical (FIG.
5N).
[0196] Besides the differences in gross clinical manifestations,
the TgH rats manifest a greater degree of AIDS-like histopathology,
such as severe splenic hyperplasia, lymphocyte depletion in lymph
nodes and apoptosis of splenocytes and endothethial cells, than do
the TgL rats. Although both TgL and TgH rat spleen show
histopathologic evidence of hyperplasia the extent of PNCA staining
is greater in the non-Tg and TgL rats than in TgH rats. The
apparently lower proliferation of splenocytes in the TgH rats
stands in contrast to their hyperplastic status. It is possible
that higher proliferation has already occurred at earlier time
points. Alternatively, it may be that the hyperplasia is a result
of an increase in cell migration to spleens in TgH rats, however
the two possibilities are not mutually exclusive and either could
occur as a result of cytokine and chemokine dysregulation by
HIV-1.
Example 8
Increased Apoptosis of Splenocytes in HIV Transgenic Rats
[0197] The occurrence of apoptosis in the HIV transgenic rats was
investigated by counting the number of Apo-Tag.RTM. positive
apoptotic cells per high powered field as follows. Five-.mu.m
tissue sections were used for in situ detection of apoptotic cells
using an Apotag kit (Oncor, Gaithersburg, Md.) and following the
manufacturer's recommendations. Spleen specimens from
five-month-old male TgH and Fisher 344/NHsd control rats were fixed
in 10% neutral formalin and embedded in paraffin. Spleens were
taken from three animals per group and each spleen section was
counted three times. Apoptotic cells in the spleen sections were
counted at 400.times. magnification using a Nikon Labophot-2 light
microscope and the apoptotic cells per high power field were
enumerated. The entire tissue section was counted using a stage
micrometer to pick successive non-overlapping fields. P-values
generated from T-test statistics with unequal variance were used to
compare mean counts of apoptotic cells per high-powered field.
[0198] The results, which are shown in FIG. 6, indicate that
apoptosis of TgH splenocytes from five month old males was
significantly elevated compared with age and sex matched
controls.
Example 9
Comparison of the Pathology of HIV Transgenic Rat with that of HIV
Infected Human Subjects
[0199] Many clinical and pathological manifestations similar to
those in AIDS in both adults and children were observed in the TgH
rats. These included wasting, neurologic changes and respiratory
difficulty. Microscopically, cardiac and renal diseases and
follicular hyperplasia of the spleen were commonly identified in
both TgH and TgL rats.
[0200] The neuropathology seen in the Tg rat is consistent with
that observed in HIV-1 infected humans. This included a reactive
gliosis, neuronal cell loss, lymphocyte infiltration and alteration
of endothelial cells with loss of blood brain barrier integrity. In
addition, there was skeletal muscle atrophy and degeneration of
peripheral nerves, similar to the human disease. The development of
a small animal model of HIV/CNS infection is likely to be of
particular utility, since the rat has been well studied in the
areas of neuroanatomy, neurophysiology, neuropathology, and
behavioral studies.
[0201] Pneumonia associated with HIV-1 infection is common in
pediatric cases of AIDS (McSherry et al. (1996) Semin. Respir.
Infect. 11:173); however, its pathogenesis is not well understood.
The most common pulmonary pathology seen in our rat model is a mild
expansion of the lung interstitium by a mononuclear infiltrate.
Most cases of pneumonia in HIV-1 infected people are associated
with secondary opportunistic infections and are characterized by
lymphocytic interstitial pneumonia. An opportunistic infection of
the lung is unlikely to be the etiology of pneumonia in our animals
because of their pathogen free status and housing conditions,
suggesting that the observed pathology is a direct result of HIV-1
expression.
[0202] A variety of cardiac disorders, including myocarditis and
cardiomyopathies, have been reported in HIV-infected patients, but
their etiopathogenesis is uncertain (Zagury et al. (1998) PNAS 95:
3851; Barbaro et al. (1998) AIDS Res. Hum. Retroviruses 14: 1071;
and Guillamon et al. (1997) Rev. Esp. Cardiol. 50:721). The gross
and microscopic cardiac pathology seen in the Tg rats is similar to
that seen in HIV-infected people and could represent a useful
noninfectious model for the study of HIV-associated cardiac
disease. There is controversy about the role of HIV as the primary
etiologic agent in HIV-1 infected people. Opportunistic infections,
cardiotoxic substances, nutritional deficiencies and autoimmune
reactions have been suggested as agents of myocardial damage. These
Tg rats are pathogen free, housed under sterile conditions and a
fed a standard certified rat diet, making it unlikely that the
observed cardiac abnormalities are due to opportunistic infections,
cardiotoxic substances or nutritional deficiencies, and suggesting
that, as with the pulmonary pathology, expression of viral gene
products is the direct cause.
[0203] Renal abnormalities in pediatric and adult AIDS, especially
in African-American patients, are common (Bourgoignie et al., Ann
Intern Med 1990 112(6):476; Guillamon, Rev. Esp. Cardiol. 50: 721
(1997)); and Bourgoignie, et al. Transplant. Proc 21: 3899-3901
(1989). The most typical renal lesions attributed to HIV-1, called
HIV-associated nephropathy (HIVAN), are seen in approximately 10-15
% of all infected adult or pediatric African-American patients.
HIVAN is a clinico-pathologic entity that includes proteinuria,
nephrotic syndrome, focal or segmental glomerulosclerosis, and
tubulo-interstitial disease, leading to a rapid progression to end
stage renal disease. Although the pathogenesis is not completely
understood, likely factors include direct effects of HIV-1 in
combination with cytokines released by HIV-1 infected or injured
cells (i.e., bFGF, TGF-beta) (Ray et al. Pediatr. Nephrol. 13:586
(1999) and Liu et al. Kidney Int. 55:1491 (1999)). Studies using
this Tg rat may provide a much-needed model that will mimic the
pathologic and clinical features of HIVAN, and will improve our
understanding of the cytopathic mechanisms of HIV-1 on different
renal cell types.
Example 10
Immunodeficiencies of the HIV Transgenic Rat
[0204] This example shows that the HIV transgenic rats have
probable antigen recognition problems. These antigen recognition
problems are manifested in an altered T-cell proliferation response
to PHA as well as a deficiency in IFN-.gamma. production secondary
to PHA stimulation. They have normal antibody production to KLH and
proliferation response to the KLH recall antigen. This data
indicates that the degree of immune deficiency is probably located
in the TH1 cells.
[0205] To determine the ability of the transgenic rats to produce
antibodies, TgH rats were injected with 100 .mu.gms of KLH in
Complete Freunds Adjuvant IP. Two weeks post-injection, the rats
were bled for antibody titer to KLH. Four weeks post-injection, the
rats were again bled for antibody titer to KLH.
[0206] To determine whether the rats have a deficiency in the T
cell compartment, delayed type hypersensitivity reactions were
conducted as follows. At four weeks, the backs of the rats were
shaved and 50 .mu.gms of KLH was given intradermally. Physical
examination of the injection site for induration of the skin in
millimeters occurred at 24 hours, 48 hours and 72 hours
post-injection. The effectiveness of the DTH response was
determined by measuring the diameter of the area of induration and
erythema. The rats were then sacrificed and T cell proliferation
assays were undertaken using KLH at 10 .mu.g/ml (recall
antigen).
[0207] The results, which are shown in Table 3 indicate that the
HIV transgenic rats (TgH rats) had a similar antibody production at
both 2 and 4 weeks and an altered DTH response, as indicated by the
smaller size of the reaction area (DTH diameter).
4TABLE 3 Anti KLH antibody titers and DTH reactions Antibody titer
Antibody titer Rats 2 weeks 4 weeks DTH diameter state control 1
1.2 .times. 10.sup.7 1.2 .times. 10.sup.7 15 mm control 2 1.7
.times. 10.sup.7 1.7 .times. 10.sup.7 15 mm control 3 2.2 .times.
10.sup.8 2.2 .times. 10.sup.8 12 mm TgH male 1 1.2 .times. 10.sup.7
1.2 .times. 10.sup.7 5 mm TgH male 2 1.3 .times. 10.sup.4 1.3
.times. 10.sup.4 <2 mm sick TgH male 3 2.7 .times. 10.sup.5 2.7
.times. 10.sup.5 ND died TgH male 4 1.8 .times. 10.sup.6 1.8
.times. 10.sup.6 ND died TgH female 1 1.7 .times. 10.sup.7 1.7
.times. 10.sup.7 5 mm TgH female 2 1.8 .times. 10.sup.6 1.8 .times.
10.sup.6 5 mm sick
[0208] In another assay, the levels of TNF-.alpha. and INF-.gamma.
were measured in TgH and control rats 4 and 7 days after
stimulation with 10 .mu.g/ml PHA. The results, which are set forth
in FIG. 7A indicated a significantly higher (at least 4 times)
production of TNF-.alpha. by the TgH rat relative to the control
rat at day 7 after stimulation. On the contrary, the production of
INF-.gamma. by the TgH rat was significantly lower at both 4 and 7
days after stimulation with PHA relative to the control rat (see
FIG. 7B).
[0209] T cell proliferation assays were performed with PBMCs from
TgH transgenic and control rats immunized with PHA. The results, of
which a representative example of an experiment is shown in FIG. 8,
indicate that the TgH rats have an abnormal proliferation response
to PHA.
[0210] Thus, the HIV transgenic rats have abnormal immune responses
characterized by an altered DTH response; a higher production of
TNF-.alpha.; a lower INF-.gamma. production; and abnormal T cell
activation (as shown here with the PHA antigen). However, the
transgenic rats have a normal proliferation response to KLH and a
normal production of antibodies (as shown here with KLH antigen
stimulation). These data strongly suggest that the immune
deficiency of the HIV transgenic rats is a Th1 immunodeficiency,
rather than a Th2 immunodeficiency, similar to what is observed in
humans infected with HIV. Thus, these results indicate that the HIV
transgenic rat can be used as a general immunodeficiency model, not
only as an HIV model.
Example 11
Production of a Human CD4 Transgenic Rat
[0211] A rat transgenic for the human CD4 gene was prepared by
inserting the human CD4 construct pLCK-CD4 described in Browning et
al. (1997) PNAS 94: 14637 (obtained from Dr. Harris Goldstein at
the Albert Einstein College of Medicine, Bronx, N.Y.). Briefly, the
construct comprises the full length coding sequence of the human
CD4 gene (described in Maddon et al. (1986) Cell 47, 333-358) under
the control of the proximal promoter for the lymphocyte specific
protein tyrosine kinase p56 Ick and 847 bp of simian virus 40
poly(A) tail coding sequence. The construct was linearized and used
to prepare a transgenic rat as described in Example 1 with the
following differences. Pseudopregnant females were obtained by
synchronysing the estrous cycle of female rats with an LH-RH
antagonist, [Ds-Gly10, D-Ala6, ProNHEt9]LH-RH. Mature SD 150-180 g
females were given 40 .mu.g of the LH-RH agonist by ip injection at
08:00 hr on day minus 4 and placed with vasectomized males on day 0
at 15:00 hr. On the morning of day 1, the females were examined for
the presence of copulatory plugs, as described above. The other
difference with the method described in Example 1 is the use of
R1ECM medium, described in Miyoshi et al. (1994) Journal of
Reproduction and Fertility 100: 21, instead of medium M16.
[0212] The huCD4 transgenic rat that was obtained was from with
bilateral small commissures. The rat was also smaller in size as
compared to a littermate control. Otherwise the rat seemed
phenotypically identical to a non transgenic rat.
[0213] Multicolor flow cytometic analysis of PBMCs isolated from
hCD4 transgenic rats was as described in Example 4 using an
anti-human CD4 antibody (commercially available from Becton
Dickinson or PharMingen). Briefly, the PMBCs were purified on
Ficoll-Hypaque and stained with PE labeled IgG1 (isotype control)
or Anti-CD4. The results, which are shown in FIG. 3, indicate that
hCD4 is expressed on PBMCs.
[0214] Expression of the transgene can be determined by PCR,
Northern blot analysis, and/or flow cytometry or FACS analysis, as
described above. PCR can be conducted using the primers described
above. The identity of the 405 fragment can be confirmed by
hybridization with an internal probe, e.g., a DNA fragment having
the sequence: 5'-GTCTCGAAGC GGGAGAAGGC GGTGTGGGTG-3' (SEQ ID NO:
10; probe CD4 1051). The absence of cross-reactivity can be
confirmed by hybridization to mouse and human RNA. For detecting
hCD4 expression, PBMC can be resuspended at a concentration of 10'
cells per ml in cold PBS with 2% serum on ice. A total of 10.sup.6
cells can then be reacted with anti-human and anti-rat CD4
antibodies (commercially available) conjugated with APC
fluorochrome and subjected to analysis by flow cytometry. It has
been observed previously that human and rat CD4 antibodies do not
cross react.
[0215] Infection of hCD4 transgenic rats with HIV can be performed
as follows. Mature (6 to 8 weeks old transgenic rats can be
inoculated either intravenously (IV) or intraperitoneally (IP) with
various concentrations of HIV (IIIB) (0.1-20 TCID.sub.50) or with
105 HIV-1 (IIIB)-infected CEM cells. Alternatively, the rats can be
infected with a T cell tropic HIV isolate. Control animals can be
non transgenic rats injected with non-infectious virus and hCD4
transgenic rats infected with the NSI HIV-1 (BA-L) or with diluted
pellets from non-infected CEM cells. The presence of HIV-1
antibodies and viral antigen (p24) in the sera can then be analyzed
every 2 weeks for the first two months and at 4 months post
inoculation using a commercially available ELISA test. Rat PBMCs
can be isolated on Ficoll-hypaque and 1.0.times.10.sup.6 cells can
be cultured with 0.3.times.10.sup.6 CEM cells. Simultaneously,
10.sup.6 PBMC can be treated with 3 .mu.g/ml of PHA overnight and
then cultured with CEM. Cultures can be examined for CPE for 1
month and supernatant can be checked for antigen production by
ELISA weekly.
[0216] DNA and RNA from the huCD4 transgenic animal can then be
extracted from PBMCs from both transgenic and non transgenic rats
and subjected to quantitative PCR for proviral DNA and multiply
spliced viral RNA, as described above.
[0217] Infection by HIV can also be tested in rats which are also
transgenic for a construct containing the HIV-1 LTR, which is
dependent on the HIV-1 Tat transactivator protein, upstream of a
reporter DNA, e.g., a gene encoding the green fluorescent protein
(GFP), for example. Infection of a cell containing this construct
with HIV will result in stimulation of the LTR and therefore the
GFP.
Example 12
Production of an HIV/Human CD4 Double Transgenic Rat
[0218] A rat transgenic for HIV, e.g, HIV-1 and human CD4 can be
prepared by crossing an HIV-1 transgenic rat described in Examples
1-10 with a rat transgenic for a human CD4 transgene, e.g.,
described in Example 11, and selecting for offspring carrying both
transgenes.
[0219] Equivalents
[0220] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents of the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
10 1 36 DNA Artificial Sequence synthetic construct 1 tagtagcatg
ctctctcgac gcaggactcg gcttgc 36 2 37 DNA Artificial Sequence
synthetic construct 2 acctcctgca gcacaggtac ccccataata gactgtg 37 3
33 DNA Artificial Sequence synthetic construct 3 atgatctgca
gttctattcc ttcgggcctg tcg 33 4 20 DNA Artificial Sequence synthetic
construct 4 agcagcagga agcactatgg 20 5 21 DNA Artificial Sequence
synthetic construct 5 ccagactgtg agttgcaaca g 21 6 25 DNA
Artificial Sequence synthetic construct 6 gagccagtag atcctagact
agagc 25 7 24 DNA Artificial Sequence synthetic construct 7
cttaggcatc tcctatggca ggaa 24 8 35 DNA Artificial Sequence
synthetic construct 8 acctcgcatg cgaagaagcg gagacagcga cgaag 35 9
20 DNA Artificial Sequence synthetic construct 9 tgacgctgac
ggtacaggcc 20 10 30 DNA Artificial Sequence synthetic construct 10
gtctcgaagc gggagaaggc ggtgtgggtg 30
* * * * *