CA2042625A1 - Transgenic non-human animal carrying a non-infectious hiv genome - Google Patents

Transgenic non-human animal carrying a non-infectious hiv genome

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Publication number
CA2042625A1
CA2042625A1 CA 2042625 CA2042625A CA2042625A1 CA 2042625 A1 CA2042625 A1 CA 2042625A1 CA 2042625 CA2042625 CA 2042625 CA 2042625 A CA2042625 A CA 2042625A CA 2042625 A1 CA2042625 A1 CA 2042625A1
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Canada
Prior art keywords
hiv
animal
transgene
proteins
dna sequence
Prior art date
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Abandoned
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CA 2042625
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French (fr)
Inventor
Paul Jolicoeur
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Institut de Recherches Cliniques de Montreal IRCM
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Institut de Recherches Cliniques de Montreal IRCM
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Application filed by Institut de Recherches Cliniques de Montreal IRCM filed Critical Institut de Recherches Cliniques de Montreal IRCM
Priority to CA 2042625 priority Critical patent/CA2042625A1/en
Priority to EP92910062A priority patent/EP0588816A1/en
Priority to PCT/CA1992/000205 priority patent/WO1992020790A1/en
Priority to AU17429/92A priority patent/AU654713B2/en
Priority to JP4509480A priority patent/JPH07500490A/en
Publication of CA2042625A1 publication Critical patent/CA2042625A1/en
Priority to US08/294,908 priority patent/US5574206A/en
Abandoned legal-status Critical Current

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Abstract

Transqenic Non-Human Animal Carrying A Non-infectious HIV Genome Abstract of the Disclosure Disclosed herein is a transgenic non-human animal carrying a transgene which expresses non-infectious HIV-1 ribonucleic acid and complementary proteins thereof. Among the expressed proteins, which can be found in the milk, serum and several tissues, are the gag and envelope proteins. The transgenic animal is useful as a source for obtaining the complementary proteins, and as an animal model to study HIV host cell interactions and to evaluate anti-HIV drugs.

Description

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Transaenic Non-Human Animal CarrYinq A Non-infectiou~ HIV Genome Field of Invention This invention relates to a transgenic non-human mamm~l carrying a transgene which expre~ses a non-infectious human immunodeficiency virus ribonucleic acid (HIV-1 RNA) and complementary proteins translated therefrom, to recombinant molecules containing the genetic sequence of the transgene, to a process for producing the transgenic mammal, and to methods of evaluating the pharmacokinetic effect of an agent on the expressed ribonucleic acid and/or proteins to determine the therapeutic value of the agent~

Backaround of the Invention In 1983, a retrovirus, known as human immunodeficiency virus type 1 (HIV-1), was e~tablished as a causitive agent of acquired immune deficiency syndrome (AIDS), see R.C. Gallo and L. Montagnier, Scientific American, 259 (4), 40 (1988). This virus has become a pestilence of alarming proportion. More recently, the closely related virus, human immunodeficiency virus type 2 (HIV-2) has been identified as a second cau~ative agent of AIDS.

The identification of human immunodeficiency virus (HIV) as a causative agent and the development of methods to grow the virus in quantity have resulted in the discovery of compounds which inhibit the replication of HIV
in vitro. The most important class of inhibitor " :~' , . :

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compounds identified in this manner is a group of dideoxynucleosides of which 3/-azido-3~-deoxythymidine (known also as zidovudine or AZT) is used therapeutically to manage certain patients with symptomatic HIV infections. This class of compound~ has been found to interfere with the life cycle of HIV by inhibiting reverse transcriptase. This enzyme converts viral RNA to double-stranded deoxyribonucleic acid (DNA~ and as such i8 an essential enzyme for HIV
replication. In addition to inhibiting reverse transcriptase, other stage~ of the HIV life cycle have been identified as targets for developing anti-AIDS drugs. One target that is receiving increaYed attention is an HIV-encoded enzyme known a~ HIV protea3e. This enzyme, like the reverse transcripta~e, is encoded by the gag-pol gene and is es~ential for HIV growth. It is responsible for effecting certain cleavages within the gag ~p55) or gag-pol (pl80) protein precur~or~ to release ~tructural proteins and enzymes, including it~elf, found in mature infectious virions. Soon after infection, the protease may cleave the core nucleocapsid, thus triggering conformational changes of the ribonucleoprotein substrate and activating DNA
synthesis. Thus, inhibitor~ of HIV protea~e may block 4everal ~tages in the HIV life cycle. For a recent review on HIV-protease inhibitors, ~ee B.M. Dunn and J. Kay, Antiviral Chemistry &
Chemotherapy, 1, 3, (1990).

Notwithstanding the progress that has been made in the cau~es and treatment of AIDS, better small animal model~ are needed to ~tudy HIV
infection~ and to evaluate potential drug~ and vaccines. The need ha~ resulted in the 204262~

development of two small animal models based on severe combined immunodeficient (SCID) mice, D.E. Mosier et al., Nature, 335, 256 (1988) and J.M. McCune et al., Science, 241, 1632 (1988).
These ~mall animal models have the potential of being used for evaluating anti-AIDS drugs and vaccine~. However, there is still a need for a small animal model to study HIV/ho~t cell interactions and to screen for anti-HIV drugs.

The present invention offers to fulfil the latter need by providing a transgenic non-human mammal, for example a mouse or a rat, which expre~ses non-infectious HIV RNA, and the structural proteins, regulatory proteins and enzyme~ translated thereform, and which can heritably transmit the transgene to it3 progeny.

The procedure for producing a transgenic animal is known in the art; for example, see B. Hogan et al., "Manipulating the Mouse Embryo:
A Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA, 1986 and T.E. Wagner and P.C. Hoppe, US patent 4,873,191, is~ued October 10, 1989. However, the prior art also teaches that it is difficult, and not precisely understood how, to obtain an animal which carries a transgene functioning in a suitable physiological environment and in a desired manner, and which can produce offspring expressing the gene. The desired gene expression can be nullified if the cloned DNA is integrated into a region of the animal's chromosome that modifies its expression, or if it undergoes mutation or rearrangement in the process of being integrated into the chromosome; see, for example, H.Van der Putten et al., Mol. Gen. Genetic, 198, ," ~ .

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128 (1984). Accordingly, a significant advance in the art of retrovirus transgenic animals, specifically HIV transgenic animals, can be realized by following the teaching of the present invention.

Previously reported production of transgenic animals include:

(a) & (b) transgenic mice containing human globin genes, T.A. Stewart et al., Science, 217, 1046 (1982) and E.F. Wagner et al., Proc. Natl.
Acad. Sci. USA, 78, 5016 (1981);

(c) transgenic mice containing the human growth hormone gene fused to a metalothionein promoter sequence; R.D. Palmiter et al., Science, 222, 809 (1983);

(d) transgenic mice with a recombinant gene containing the rat elastase I promoter and the f codons for human growth hormone, D.M. Ornitz et al., Nature, 313, 600 ~1985);

(e) transgenic mice containing the human insulin gene, R.F. Selden and H.M. Howard, PCT patent application WO 87~07298, published December 3, ; 1987;

(f) several transgenic mice cited in a review by G. Skangos and C. Bieberich, Advances in Genetics, 24, 285 (1987);

(g) several transgenic mice cited in a review by R.M. Strojek et al. entitled "The Use of Transgenic Animal Techniques for Livestock Improvement" in "Genetic Engineering: Principles - ~ .

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and Methods", J.K. Setlow, Ed., Vol. 10, New York, NY, USA, 1988;

(h) transgenic non-human animals having a tran~gene with an activated oncogene sequence, P. Leder and T.A. Stewart, US patent 4,736,866, i~ued April 12, 1988;

(i) several tran~genic animal~ cited in a review by J. Van Brunt, Bio/Technology, 6, 1149 (1988);

(j) transgenic animals, containing hormone encoded gene~, from which hormones can be harve~ted, R.M. Evans et al., US patent 4,870,009, issued September 26, 1989;

(k) transgenic animals containing a gene capable of producing recombinant proteins in the animal's milk, H. Meade and N. Lonberg, US patent 4,873,316, i~ued October 10, 1989; and (l) tran~genic mice, useful for analyqi~ of high growth, having recombinant genes comprising the regulatory element~ involved in the expre~sion of hair ~pecific gene~, A.R. McNab et al., Canadian patent application 2004156, publi~hed May 31, 1990.

Previous reports of tran~genic mice carrying all or part of the HIV-1 genome include:

(a) a line of transgenic mice carrying only the long terminal repeat (LTR) of HIV fused to the chloramphenicol acetyl tran~ferase (CAT) reporter gene, which were mated to tran~genic mice of the opposite sex carrying the HIV-1 tat gene fused to a control element of the murine A~ crystallin gene to give progeny carrying both gene~; hence, only a small portion of the HIV-l genome is involved, J.S. Khillan et al., Nucleic Acid~
Res., 16, 1423 (1988);

(b) transgenic mice having the HIV-l tat gene linked to the HIV-l LTR in the absence of other parts of the HIV-l genome and which developed skin lesions resembling Xaposi's sarcoma, J.
Vogel et al., Nature, 335, 606 (1988);

(c) a ~ingle founder line of transgenic mice carrying a complete HIV-l proviral genome wherein the off~pring developed a disea~e ~yndrome resembling some aspects of the symptoms of AIDS
patients, and marked by the pre~ence of HIV in the tissues of the offspring and premature death thereof, J.M. Leonard et al., Science, 242, 1665 (1988);

(d) four lines of transgenic mice containing the HIV-l LTR linked to the CAT reporter gene but no other parts of the HIV-l genome and for which CAT
activity wa~ observed among mononuclear cells and maximally in Langerhans~ cells, J. Leonard et al., AIDS Res. Hum. Retrovirus, 5, 421 (1989);

(e) trangenic progeny from three founder mice bearing chromosomally integrated copies of partial HIV-l proviral DNA (rendered non-infectious by deletion of the gag-pol sequences) and which developed glomerulosclerosis, P. Dickie et al., Sixth International Conference on AIDS, San Francisco, Vols 1-3, abstract Th.A. 290 ( 1990 );

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(f) transgenic mice carrying HIV TAT and NEF
genes under control of the HIV LTR, but no other parts of the HIV genome, as well as two transgenic founders carrying the HIV NEF gene construct (without TAT), J. Dinchuk et al., Sixth International Conference on AIDS, San Franci~co, Vols. 1-3, abstract Th.A. 291 (1990);

(g) transgenic mice bearing the LTR of HIV (but no other part~ of the HIV-1 genome) fused to the reporter gene CAT, one line of which showed CAT
activity in the cerebellum, E. Harlan and O.P. Rakash, Society for Neuro~cience Ab~tract~, Vol. 16, 353 (1990), abstract 154.1; and (h) three lines of transgenic mice carrying a partial HIV genome comprising the HIV-l LTR fu~ed to the ~imian virus 40 early region, for which the transgene was observed in the lymphoid tis~ue and ~kin of the mice, J. Skowronski, J. Virol., 65, 754 (1991).

The transgenic animal of the present application is di~tinguished from the transgenic animals of the prior art in that it carries a non-infectious transgene having the complete coding sequence of the HIV genome, the transgene being capable of producing high levels of HIV
protein~ in ~everal tis~ues and certain body fluids of the animal. The attainment of the pre~ent transgenic mammal is surpri~ing indeed in view of previous reports that expression of HIV
protein~ wa~ found to be very low in mouse fibroblasts transfected with the HIV genome; for example, see J.A. Levy et al., Science, 232, 998 (1986).

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Summary of the Invention The present invention provides a non-human transgenic mammal in which the germ cells and somatic cells carry a tran~gene capable of expressing non-infectious HIV-l ribonucleic acid (HIV-1 RNA) and complementary proteins in the cells. The tran~gene i~ introduced into the animal, or an ancestor of the animal, at an embryonic stage, preferably between the one-cell (or fertilized oocyte) and the 8-cell stage.

Among the expre~sed complementary proteins, which can be found in the milk, serum and several tissues of the transgenic animal are the gag and envelope proteins.

A noteworthy feature of the pre~ent transgenic animal is that it can produce progeny in which the gene is stably expressed. Hence, animal lines of the transgenic animal, having the same e~sential characteristics, are realized.

The transgene of the transgenic animal of this invention comprises (a) a non-infectious HIV-1 proviral DNA ~equence, (b) a surrogate promotor DNA sequence operatively linked to a tissue-specific DNA sequence to provide a surrogate promoter/tissue-specific enhancer, which in turn is operatively linked to the proviral DNA sequence, and (c) at least one polyadenylation addition signal DNA sequence operatively linked to the proviral DNA sequence.

A preferred embodiment of the transgene for use in the animal comprises (a) a proviral DNA
sequence, coding for HIV-l RNA, in which the 5/-204262~

long terminal repeat (5~-LTR) sequence, a portion of the 5/ leader sequence and a portion of the 3/-long terminal repeat (3/-LTR) have been deleted, the deleted portions being predetermined to render non-infectious any HIV-l RNA and complementary proteins expres~ed within the cells of the animal; (b) a surrogate promoter~tissue-specific enhancer DNA sequence operatively linked to the S/-end of the proviral DNA sequence to drive the RNA expre~sion of the transgene in A
preselected tissue of the animal; and (c) one or more polyadenylation (poly A) addition signal sequences, operatively linked at the 3/ end of the proviral DNA to provide 3/ maturation and stabilization of the expressed RNA.

In a more preferred embodiment of the tran~gene, the surrogate promoter/tis~ue-specific enhancer ~equence i8 the LTR promoter of the mou~e mammary tumor virus ~MMTV), al80 known a~
MMTV LTR, and the poly A addition ~ignal sequence i~ the SV40 poly A addition ~ignal.

Disclosed hereinafter are a procesY for preparing the aforementioned transgene from a recombinant DNA plasmid in which the DNA sequence of the tran~gene has been incorporated therein, and in turn a process for preparing the plasmid.
The processes and the plasmid are included within the scope of thi~ invention.

Also included within the scope of the invention is a method of producing a tran~genic animal, carrying the aforementioned transgene, which can be stably bred to produce offspring containing the gene. The method comprises:

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20~2625 (a) i~olating a fertilized oocyte from a fir~t female animal;

~b) transferring the transgene into the fertilized oocyte;

(c) tran ferring the fertilized oocyte containing the transgene to the uterus of the same species a~ the first animal;

(d) maintaining the second female animal ~uch that (i) the ~econd female animal become~
pregnant with the embryo derived from the fertilized oocyte containing the transgene, (ii) the embryo develops into the tran~genic animal, and (iii) the transgenic animal i~ viably born from the ~econd female animal;

wherein the transgenic animal ha~ the genetic sequence for non-infectiou~ HIV proviral DNA and i~ capable of being bred to produce offspring having cell~ ~tably containing the genetic sequence.

Noteworthy is that by monitoring the effect of a test compound on the level~ of expre~ed non-infectiou~ RNA, and the translated proteins therefrom, e.g. the gag and envelope protein~ and the gag protein cleavage products, the therapeutic value of a te~t compound for treating HIV infection~ in humans can be evaluated.

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For in~tance, the proteins expres~ed in the cells of the transgenic animal include proce~sed 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, the invention also provides a method for evaluating a te~t compound as a potential HIV-1 protea~e inhibitor. The method involves:

(a) administering the test compound to the transgenic animal, and (b) examining the effect or significance of the test compound on the expressed gag proteins in the animal by monitoring the expression levels of the proteins.

Likewise, since the presence of the gag and envelope protein~ in the fluid and tissues of the transgenic animal denotes that the HIV regulatory protein, REV, is expres~ed, the pre~ent invention provides a method for evaluating a test compound as a potential inhibitor of REV function. The method involves:

(a) administering the test compound to the transgenic animal, and (b) examining the effect or significance of the te~t compound on the expres~ed gag and envelope proteins and the gag protein cleavage products in the animal by monitering the expression levels thereof.

Still another aspect of this invention involves the use of the transgenic animal for the production and isolation of the non-infectiou~
RNA and its complementary proteins.

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De~criPtion of the Drawin~s Figure 1 is a diagrammatic representation of a region of a pla~mid bearing the non-infectiou~
HIV proviral DNA ~equence and flanking region~.
The re~triction isite~ are A, AatII; B, ~amH1; E, EcoR1; H, HindIII; S, SacI; and X, XbaI.

Figure 2 i8 a reproduction of a photograph of a Western blot analysis of human T-cells infected with HIV-1 (lane 1), milk from representative female transgenic mice of thi~
invention (lanes 2 and 4) and milk from normal C3H mice (lanes 3 and 5). The HIV gag proteins are similarly processed in human T-cells and in the transgenic mouse. No HIV-specific proteins are detected in the milk of the normal mouse.

Detail~ of the Invention The term "HIV" and "HIV-l" are used interchangably herein and refer to the human immunodeficiency viru~ type 1. It should be noted, however, that the technology of the present application can be adapted to produce a transgenic animal carrying a transgene expres~ing HIV-2 RNA. Accordingly, the latter transgenic animal is deemed to be within the scope of this invention.

The term "gene" as used herein means the smallest, independently functioning DNA sequence which encodes for a protein product. An example of a gene is the DNA isequence which encodes for the gag protein.

20~2625 The term "tran~gene" a~ u~ed herein mean~
exogenous genetic material which does not naturally form part of the genetic material of an animal to be genetically altered but can be incorporated into the germ and ~omatic cells of that animal by standard tran~genic technique~.
The transgene of the present invention is created by the serial ligation of a promoter DNA ~egment, a major portion of the HIV-1 genome including the coding region for gag-pol and envelope, and at least one poly A addition DNA sequence.

The term "operatively linked" as used herein in reference to units (i.e. distingui~hable DNA
sequences) of a transgene, means that the units to which the term is applied are linked according to recombinant technology techniques 80 that they may act together to control and expres~ the transgene encoded RNA in a suitable tissue or cell type. An example would be the operatively llnking of a promotor/tiesue-~pecific enhancer to a DNA sequence coding for the desired protein~ 80 as to permit and control expression of the DNA
sequence and the production of the complementary proteins.

The term "prcmoter" as used herein means a DNA sequence which binds RNA polymerase and directs the enzyne to the correct transciptional start site. The promoter is upstream of the HIV
proviral DNA sequence to which it is operatively linked.

The term "enhancer" as used herein refers to a DNA sequence capable of increasing the tran~cription of the promoter. The term "promoter/tissue-specific enhancer" means a DNA

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sequence, having an activation site which is capable of being activated in a specific tissue or specific cell type, to promote the preferential transcription of a given gene to which the DNA sequence i8 operatively linked.
Thus the tran~ciption and expre~sion of the tran~gene having the promoter/ti~sue-specific sequence will be enhanced within the specific tissue or cell-type. Examples of promoter/tissue-specific enhancers are the aforementioned MMTV LTR and the promoter/brain-specific enhancer, the myelin basic protein (MBP) promoter.

The term "complementary proteins n or "associated proteins" as used herein in relation to HIV-l RNA refers to the initially translated proteins and their sub~equent cleavage products.
In particular, the term refers to the readily isolated and detectable proteins such as the HIV-1 gag protein (p55), the HIV-l gag protein cleavage products p24 and pl7, the envelope glycoprotein gpl60 and the envelope protein cleavage product gpl20.

A~ indicated previously, procedures for producing transgenic animals have advanced considerably during the last ten years. This advance has provided new avenues for seeking improvement and creative applications. A
compendium of the techniques can be found in the textbook by B. Hogan et al. entitled "Manipulating the Mouse Embryo: A Laboratory Manual", Cold Spring Har~or Laboratory, Cold Spring Harbor, NY, USA (1986). The latter reference is incorporated herein by reference.

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More explicitly with regard to the present invention, the key feature is the realization of an animal carrying a transgene expressing non-infectious HIV-l RNA, which in turn is tran~lated 5to give complementary HIV proteins in its body fluids and tissues.

Any mammal may be used in the production of the transgenic animal of this invention. The choice of animal will depend on the particular 10use planned for the transgenic animal.

When it is envisaged to u-~e the tran~genic animal as a del to study or evaluate the effects of a test compound on the expression of the HIV RNA and its complementary proteins, the 15practical choice of animal i~ one that is ~mall, inexpensive and breeds readily and rapidly. In this cAse, the practical choice i~ a rodent, preferably a rat or a mouse. As noted in B. Hogan et al., supra, the mouse has been the 20favored small animal for the development and applications of transgenic technology; however the rat is also a practical animal species for this purpose; for example, see R.E. Hammer et al., Cell, 63, 1099 (1990).

25When it is envisaged to use the transgenic animal as a source of HIV-l RNA and its complementary proteins, i.e. for the production of research quantities of the non-infectious RNA
transcript and associated proteins, e.g. gpl60, 30gpl20, p55, p41, p24, pl7, REV, etc., a larger animal can be used. A convenient and practical proces~ for such production purposes compri~e~
the use of mammals ~hat have long lactating period~ and produce large volumes of milk; for ~; , , - ~ ..

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example cow~, ~heeps, goat~ and pigs, which are genetically altered with a transgene of this invention ~o that the desired products are secreted into the mammal's milk. In this instance the transgene comprise3 a promoter/tissue-specific enhancer which is a mammary tissue specific promoter, or any promoter sequence known to be activated in mammary tissue, operatively linked to the non-infectious HIV
proviral DNA sequence. The desired products are i~olated from the collected milk of the animal by ~tandard procedures.

More explicitly with regard to the construction of the present recombinant tran~gene, the transgene comprises three fragments: firstly, a proviral DNA sequence having deleted portions at it~ 5/ and 3/ ends 80 as to render non-infectious the expressed HIV RNA
and complementary proteins; secondly, a surrogate promoter/tis~ue-specific enhancer DNA sequence to drive the expression of the proviral DNA
sequence; and thirdly, one or more p41y A
addition signal DNA 8equence8.

The promoter/ti~sue-specific enhancer i9 located upstream from the proviral DNA 8equence;
and the poly A addition signal sequence is located downstream of the proviral DNA sequence.

The HIV proviral DNA sequence i~ prepared by dige~ting a plasmid clone containing the DNA
sequence of HIV-l with a restriction enzyme that cleaves the HIV proviral DNA sequence at sites proximal to its 5/ and 3/ end~, removing essential controling sequences, t4 give a proviral DNA
~equence truncated at both end~ 80 that the :

eventual RNA expres~ion from the cleaved fragment is rendered non-infectious, but still includes those elements required for the eventual production of the complementary proteins. In other words, the HIV genome lacks the ~equencefi necessary for reverse transcription, integration and transcription. [The extent to which the 5/
and 3/ ends must be truncated to render the RNA
non-infectious can be determined by ~tandard methods; for example, by transforming the fragment 80 obtained into a genomic equivalent of HIV-1 and testing the resulting virus for cytopathic activity.~ For example, a particular plasmid and restriction enzyme for effecting this result is the pBH10 plasmid [B.H. Hahn et al., Nature, 312, 166 (1984)1 and the restriction enzyme SacI. The latter restriction enzyme cleaves the plasmid to give an HIV-1 genome deleted of its 5/ LTR, part of its untranslated 5 leader sequence and a portion of its 3/ end LTR.

Concerning the surrogate promoter/tissue-specific enhancers useful in various embodimQnts of this invention, any such promoter/enhancer which effectively drives the expression of the non-infectious HIV genome can be used. The choice of the pro ter/enhancer depends on the tissue or tissues in which it is desired to express the HIV RNA and associated proteins. For instance, if it is desired to express the desired product~ in the milk, promoters which are known to be activated in the mammary gland are employed. Examples of ~uch milk specific protein promoters are the casein promoter~ and the ~-lactoglobin promoter. Among the promoters that are specifically activated in the milk and thus useful for expressing the desired products in .

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milk according to the present invention i~ the MMTV LTR. By way of another example of promoters, when it i8 de~ired to specifically express the non~infectious RNA and associated protein~ in the brain of the transgenic animal, promoterq which are known to be activated in the brain are incorporated into the transgene. Such animal~ provide a useful in vivo model to evaluate the ability of a potential anti-HIV drug to cros~ the blood-brain barrier. An example of brain-specific protein promotex is the myelin basic protein (MBP) promoter.

In a preferred embodiment of the transgene, the ~urrogate promoter/tissue-specific enhancer to drive expression of the HIV genome is the mouse mammary tumor virus long terminal repeat (MMTV LTR) sequence. Thi~ promoter i9 known to be ti~ue ~pecific toward variou~ epithelial and hemopoietic ti~sues, ~ome of which naturally support lentiviru~ (and especially HIV) replication.

Concerning the poly A addition signal ~equence, this fragment i8 one or a tandem of-two to four of the known poly A addition ~ignal sequences, ~uch a~ 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 ~equence can be cleaved.

The transgene can be prepared from the aforementioned three fragment by using techniques known in the art; for example see J. Sambrook et 204262a al., "Molecular Cloning: A Laboratory Manual", 2nd ed, Vols 1 to 3, Cold Spring Harbor Laboratory Pre~, Cold Spring Harbor, NY, USA, 1989, herein incorporated by reference. More ~pecifically, the three fragments can be ligated together with T4 DNA ligase in the presence of a restriction endonucleas-dige~ted vector to produce the gene-carrying plasmid with the desired orientation of the fragments, i.e. ~o that they are operatively linked. Subsequent cleavage of the latter pla~mid at appropriate unique siteY i8 effected by re~triction endonuclease~ to excise the linear tran~gene, which after appropriate purification is incorporated into the animal to be genetically altered.

The linear transgene (hereinafter sometimes referred to as the MMTV/HIV fragment) can be added to the gene pool of the animal to be altered by incorporating one or more copie~ of the transgene into the genome of a mammalian embryo by techniques known in the art. A
practical technique is to introduce the transgene at the embryonic ~tage of the animal by microinjecting the transgene into a fertilized oocyte of the animal between the one-cell and eight-cell ~tage, preferably between the one-cell and four-cell stage. In this manner, between 5 to 30% of the animals developing from ~uch oocytes contain at least one copy of the transgene in somatic and germ cells, and these founder animals transmit stably the transgene through the germ line to the next generation.

The biological profile of the pre~ent tran~genic animal renders it useful a an animal ' ~

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model for ~tudying HIV/host cell interactions and for evaluating potential anti-HIV agents acting at a po~t-integration step of the HIV life cycle.
On the ba~i~ that the main structural gag proteins, e.g. p55, p41 and p24, are expre~sed in the animal cells, potential HIV-1 protease inhibitors can be evaluated since the HIV
protease is essential for the processing of mature proteins from the gag-pol polyprotein.
The presence of the RNA transcript and the presence or decrease (or disappearance) in the proces~ed proteins in the cell~ serve~ a~ a meanY
for evaluating HIV protease inhibitors.

On the basis that the main structural HIV
protein~ are properly made, e~pecially the gag and envelope proteins and the gag cleavage products, potential REV function inhibitor~ can be evaluated ~ince the REV function i8 essential for the production of tho~e proteins. Thus, the monitoring of the levels of the latter proteins in the cells of the transgenic animal receiving a test agent provide~ a means for evaluating the anti-REV function activity of the agent.

Likewise, a potential inhibitor of cellular protease required for the processing of envelope glycoproteins, can be detected by monitoring the levels of gag and envelope proteins. In this in~tance, the cellular protease inhibitor does not affect the cellular levels of gag proteins but it does decrease the levels of the mature envelope glycoproteins, e.g. gpl20, or cause their disappearance from the body cells. Thus, the comparative effect~ on the translation of gag and envelope proteins ~erve~ as an indication of cellular protease inhibition.

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The following examples further illu~trate this invention.

ExamDle 1 Construction of Transaene (12 kbD MMTV/HIV fraament) The transgene con~i~ted of three fragments;
the MMTV LTR promoter/tissue-~pecific enhancer sequence, the HIV proviral DNA sequence and the poly A addition signal sequence. The fragments were prepared as follows:
(a) MMTV LTR promoter sequence - The 2.3 (kbp) BamH1 MMTV fragment containing the 3/ end sequences of the MMTV genome (including the 3 env sequences with the whole 3/ end LTR) was obtained by digestion of plasmid pA9, A.L. Huang et al., Cell, 27, 245 (1981), followed by subcloning of the resulting fragment at the BamH1 site of plasmid pUC18 in an orientation which placed the end of the LTR close to the EcoR1 site. It was then prepared as a HindIII - SacI
fragment by digestion with the restriction enzyme HindIII and then a partial digestion with the restriction enzyme SacI. Thus both of the enzymatic sites of the fragment were derived from the polylinker of the vector.

(b) HIV proviral DNA sequence - The 8.9 kbp SacI
HIV-1 fragment was obtained by digesting the pla~mid pBH10, B.H. Hahn et al., Nature, 312, 166 ~1984), with SacI.

(c) poly A addition signal sequence - The SV40 poly A addition signal was prepared by cleaving the 885 bp BamH1 - PstI fragment from the pSV2neo vector (ATCC 37149) and subcloning the fragment .

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80 obtained in the plasmid pUC18 (ATCC 37253).
The SV40 fragment was then prepared from the subclone by digeffting the subclone with SacI and HindIII.

U~ing T4DNA liga~e, the three fragment~ from sections (a), (b) and (c) were ligated together in a HindIII-dige~ted pBR322 vector in an orientation which placed the 3/ end SV40 sequences close to the EcoRl site of the plasmid pBR322. (See J. Sambrook et al., supra, section 1.12, for a description of p~R322.) In thi~
manner, the gene-carrying pBR322 plasmid, incorporating the transgene DNA sequence (i.e.
the 12 kbp MMTV/HIV fragment), was obtained.
Thi~ plasmid is diagrammatically represented by Figure 1.

The 12 kbp MMTV/HIV fragment for microin~ection, see example 2, was obtained by cleaving the gene-carrying pBR322 plasmid with restriction enzymes XbaI and AatII. The fragment was isolated by agaro~e gel electrophoresis and further purified on CsCl gradients, essentially as described by B. Hogan et al., supra~ p 159.
In thi~ manner, the 12 Kbp MMTV/HIV fragment was obtained wherein the proviral DNA sequence therein lacks the 5/-LTR sequence, a portion of the 5/-leader sequence and a portion of the 3~-LTR, thus rendering the fragment non-infectious.

ExamPle 2 Con~truction of Tran~qenic Mice One-cell (C57BL/6 x C3H)F2 embryos were collected, microinjected with the 12 Kbp ~MTV/HIV
fragment of example 1 (weight concentration = ca .

, . -.
' 20~2625 1 ~g/mL) and tran~ferred into p~eudopregnant CD-1 female~ e~sentially a~ de~cribed before [Hogan et al., supr~ and L. Bouchard et al., Cell, 57, 931 (1989)l. From 85 eggs, microin~ected w~th the fragment and reimplanted, 17 mice were born, four of which were later found to be transgenic by Southern blot analysis of tail DNA with a 32p_ HIV-1 qpecific probe (~ee J. Sambrook et al, supra, ~ection~ 9.31 to 9.57).

The HIV-~pecific probe was prepared by labeling the aforementioned 8.9 kbp SacI HIV-l fragment with 32P-deoxycyto~ine tripho~phate and deoxyadenosine tripho~phate by the random primer method (~ee J. Sambrook et al., supra, ~ection 10.13).

In each of the four tran~genic mice, the MMTV/HIV ~equence~ appeared intact and localized at an unique integration ~ite (data not ~hown).

Three of the tran~genic mice were bred successfully to C3H mice, obtained from Charles River Laboratories Canada Inc., St-Con~tant, Québec, Canada. The~e three founders tran~mitted the transgene to their progeny in a Mendelian fa~hion. The three line~, i.e. MMTV/HIV-R3, MMTV/HIV-R4 and MMTV/HIV-R10, were thu~
e~tabli~hed by mating founder mice to C3H mice.

- ~ . . ~ , .

. .
.

20~262~

Exam~le 3 Transqenic Mouse Studies (a) PhenotYDe of mice carrYina the MMTV/HIV
transaene No phenotype was apparent in the transgenic mice. They looked, behaved and moved like the age-matched, non-transgenic mice. At autopsy, macroscopic examination of the tran~genic mice indicated that they were normal.

(b) RNA transcriDt HIV-1 RNA expression of the transgene wa~
detected by the agarose gel transfer (Northern) procedure, using total RNA from variou~ organ~ of representative transgenic mice.

More particularly, the total RNA was isolated by the method of P. Chomczynski and N.
Sacchi, Anal. Biochem., 162, 156 (1987) and analyzed by the Northern blot procedure (see J.
Sambrook et al., supra, section 7.39). RNA was separated on 1% formaldehyde-agarose gels, transferred to nylon membranes (Hybond-N~, Amersham Canada Ltd., Oakville, Ontario, Canada) and hybridized with the aforementioned 32P-HIV-specific probe according to the method of L. Singer and K.W. Jone~, Nucl. Acids Res., 12, 5627 (1984).

The three main ~pecies of HIV-1 RNA
(classified according to their molecular weight), ~; usually detected in cell~ productively infected with HIV-1, were detected in several organs known to support transcription from the MMTV LTR, such ... .

.

204262~

as mammary, Harderian and salivary glands, epididymis, thymus and ~pleen, in the three transgenic lines te~ted. Additional investigations were carried out on other organs S of the mice of the line NMTV/HIV-R10. In those organ~ which are known to ~upport MMTV-LTR-driven transcription poorly, such as the kidney, liver or brain, the examination of the organ~ for transgene RNA transcript indicated that the transgene was not expre~sed, as expected.

(c) Exression of HIV structural Protein in oraans of the transqenic mice.

To determine whether the HIV-l RNA found in organs of the transgenic mice wa~ tran~lated, the levels of HIV structural proteins in these organs were measured by the Western blotting procedure (see E. Harlow and D. Lane, "Antibodies : A
Laboratory Manual", Cold Spring~ Harbor Laboratory, Cold Springs Harbor, NY, USA, 1988, section 12) with antisera specific to HIV-l proteins. The env gpl60 and gpl20 were detected at very high or moderate levels in the mammary gland~ of animals of three transgenic lines, at lower levels in the salivary and Harderian glands, and in the liver, seminal ve~icles, epididymis and testis of mice from the line MMTV/HIV-R10, and in the epididymis, spleens or livers of mice from line MMTV/HIV-R4. These proteins comigrated with authentic HIV env proteins from HIV-l infected cells, except that the transgenic gpl20 migrated slightly faster.

The gag p55 precursor protein was detected at high levels in mammary glands of animals of the three transgenic lines. The p55 and/or p41 204262~

protein~ were detected in the salivary and Harderian glands, and in the spleen, seminal ve~icles and epididymis of mice from line M/HIV-R10 and in the ~pleen and epididymi~ of mice from line MMTV/HIV-R4. The mature cleavage product, the p24 gag protein was detected in mammary glands of mice from line MMTV/HIV-R10 and MMTV/HIV-R3 and in the ~alivary glands of animals from line MMTV/HIV-R10. The pS5, p41 and the p24 proteins from the above designated organ~ of the tran~genic mice migrated with corre~ponding authentic protein~ from HIV-1 infected human T-cell~, indicating proper synthe~is and cleavage in the~e mou~e organ~.

In serum, HIV-1 proteins reactive with HIV-~pecific antibodies were detected in mice from two lines ~MMTV/HIV-R4 and MMTV/HIV-R10). Higher level~ of HIV-1 ~erum protein~ were detected in lactating female mice.

The HIV env (gp 160 and gp 120) and gag (p55, p41 and p24) proteins were also detected at very high levels in milk from lactating female tran~genic mice (see Figure 2). In addition, HIV
protein~ were detected at a lower level in the secretions of the male genital tract. These reqult~ were expected since the mammary gland~ aY
well a~ the epididymi~ were positive for HIV-l proteins, see above.

Th2se re~ult~ clearly indicated that several mouse specialized cell types are capable of releasing HIV proteins extracellularly either in the circulation or in ~ecretions.

::
. ~

` 20~2625 Search for the Presence of MuLV(HIV~ or HIV~MuLV) PseudotY~es in serum of transaenic mice.

HIV and murine leukemia viruses (MuLV) have been ~hown to form pseudotypes under certain experimental conditions. Although a short leader sequence necessary for HIV RNA packaging is still present in the transgene, the HIV RNA sequence~
transcribed in these mice would not be expected to be packaged very efficiently, due to the absence of other leader and LTR sequences.
Moreover, if packaged, these sequences would not be expected to be reverse transcribed, becau~e of the absence of U5, R5 and part of the U3 and R3 sequences of HIV-l. HIV proteins, however, should be able to form pseudotype~ with MuLV
proteins and may be able to package endogenous MuLV RNA.

To detect infectious particles having a MuLV
RNA genome and HIV env proteins, A3.01 cells, known to be resistant to ecotropic and xenotropic MuLV infection but ~ensitive to ~uLV replication and harboring the CD4 receptor for HIV, were used. These human cells, possibly infected.and producing MuLV, were cocultivated with NIH/3T3 or . 25 mink cells, respectively susceptible to ecotropic or xenotropic MuLV. No such MuLV (HIV) ecotropic or xenotropic pseudotype was detected in serum of the tran~genic mice from lines MMTV/HIV-R4 and MMTV/HIV-R10 indicating that these pseudotypes were ab~ent or below the sensitivity of the assay.

Therefore, it appears that the formation of MuLV (HIV) pseudotypes is not characteristic of these transgenic mice.

In conclusion, the transgenic animal of the invention therefore provides a ~afe and practical model to ~tudy the effects of test compound~, when administered ~ystemically to the animal, on the expressed HIV RNA, proce~ed gag protein~, the pol encoded enzymes, envelope proteins and regulatory proteins (e.g. REV ) . For in~tance, the readily measurable high levels of the HIV-l gag reactive proteins, p55, p41 and p24, in the milk of the lactating female transgenic mice, noted above, renders the latter animal a convincing and practical model for evaluating a te~t compound as a HIV-l protease inhibitor by admini~tering the te~t compound, po, sc, ip or iv, to the animal, and monitoring the effect of the test compound on the expression levels of the HIV-1 gag reactive proteins in the milk of the animal, for example, by the Western blotting procedure utilizing a gag p24 or pl7 specific antibodies.

Claims (14)

1. A non-human transgenic mammal in which the germ cells and somatic cells carry a transgene capable of expressing non-infectious HIV-1 RNA
and complementary proteins in the cells, the transgene being introduced into the mammal, or an ancestor thereof, at an embryonic stage.
2. The mammal of claim 1 wherein the transgene comprises (a) a non-infectious HIV-1 proviral DNA
sequence, (b) a surrogate promoter/tissue-specific enhancer operatively linked to the proviral DNA sequence, and (c) at least one poly A addition signal DNA sequence operatively linked to the proviral DNA sequence.
3. The mammal of claim 1 wherein the transgene comprises:

(a) a proviral DNA sequence, coding for HIV-1 RNA, in which the 5'-LTR sequence, a portion of the 5' leader sequence and a portion of the 3'-LTR have been deleted, the deleted portions being predetermined to render non-infectious any expressed HIV-1 RNA or complementary proteins thereof;

(b) a surrogate promoter/tissue-specific enhancer DNA sequence operatively linked to the 5'-end of the proviral DNA sequence to drive the RNA expression of the transgene in a preselected tissue of the animal; and (c) one or more poly A addition signal DNA
sequences, operatively linked at the 3' end of the proviral DNA sequence to provide 3' maturation and stabilization of the expressed RNA.
4. The mammal of claim 2 wherein the surrogate promoter/tissue-specific enhancer is the MMTV LTR
promoter and the poly A addition signal sequence is the SV40 poly A addition signal.
5. The mammal of any one of claims 1 to 4 wherein the mammal is selected from a cow, sheep, goat, pig, rat and mouse.
6. The mammal of any one of claims 1 to 4 wherein the mammal is a mouse.
7. The recombinant transgene as recited in any one of claims 2 to 4.
8. A process for preparing a transgene comprising the following three DNA sequences: (a) a proviral HIV DNA sequence having a deleted portion at its 5' and 3' ends so as to render non-infections expressed HIV RNA and complementary proteins therefrom; (b) a surrogate promoter/
tissue-specific enhancer DNA sequence, operatively linked to the 5'-end of the proviral DNA sequence; and (c) one or more poly A addition signal DNA sequences operatively linked to the 3' end of the proviral DNA; said process comprising;
ligating in the desired orientation the three DNA
sequences into a restriction endonuclease-digested vector to obtain the corresponding gene-carrying plasmid and cleaving the plasmid at unique sites with restriction endonucleases to excise the transgene.
9. A gene-carrying plasmid when prepared by ligating in the desired orientation the three fragments of claim 8 into a restriction endonuclease-digested vector.
10. A gene-carrying plasmid when prepared by ligating in the desired orientation the three fragments as defined in claim 8 into a HindIII-digested pHR322 vector.
11. A method for producing a transgenic mammal of claim 2, comprising:

(a) isolating a fertilized oocyte from a first female animal;

(b) transferring the transgene of claim 2 into the fertilized oocyte;
(c) transferring the fertilized oocyte containing the transgene to the uterus of the same species as the first animal;

(d) maintaining the second female animal such that (i) the second female animal becomes pregnant with the embryo derived from the fertilized oocyte containing the transgene, (ii) the embryo develops into the transgenic animal, and (iii) the transgenic animal is viably born from the second female animal;
wherein the transgenic animal has the genetic sequence for non-infectious HIV-1 proviral DNA and is capable of being bred to produce offspring having cells stably containing the genetic sequence.
12. A method for evaluating a test compound as a potential HIV-1 protease inhibitor, comprising:

(a) administering the test compound to the transgenic animal of claim 1, and (b) examining the effect or significance of the test compound on the expressed gag proteins in the animal by monitoring the expression levels thereof.
13. A method for evaluating a test compound as a potential inhibitor of REV function, comprising:

(a) administering the test compound to the transgenic animal of claim 1, and (b) examining the effect or significance 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.
14. A process for the production of non-infectious HIV-1 RNA or the complementary proteins thereof, comprising:

a) collecting the milk from the transgenic animal of claim 2 wherein in the promoter/tissue-specific enhancer DNA sequence is a mammary tissue specific promoter;
b) collecting the milk, and c) isolating the non-infectious HIV-1 RNA, or the complementary proteins thereof, from the milk.
CA 2042625 1991-05-15 1991-05-15 Transgenic non-human animal carrying a non-infectious hiv genome Abandoned CA2042625A1 (en)

Priority Applications (6)

Application Number Priority Date Filing Date Title
CA 2042625 CA2042625A1 (en) 1991-05-15 1991-05-15 Transgenic non-human animal carrying a non-infectious hiv genome
EP92910062A EP0588816A1 (en) 1991-05-15 1992-05-14 Transgenic non-human animal carrying a non-infectious hiv genome
PCT/CA1992/000205 WO1992020790A1 (en) 1991-05-15 1992-05-14 Transgenic non-human animal carrying a non-infectious hiv genome
AU17429/92A AU654713B2 (en) 1991-05-15 1992-05-14 Transgenic non-human animal carrying a non-infectious HIV genome
JP4509480A JPH07500490A (en) 1991-05-15 1992-05-14 Transgenic non-human animal carrying a non-infectious HIV genome
US08/294,908 US5574206A (en) 1991-05-15 1994-08-24 Transgenic mouse carrying a non-infectious HIV genome

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CA 2042625 CA2042625A1 (en) 1991-05-15 1991-05-15 Transgenic non-human animal carrying a non-infectious hiv genome

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