This invention provides cellular gene products which have anti-apoptotic activity in HIV-1 infected cells and provides agents for the inhibition of the cellular gene products.

Description of the Invention

SUMMARY OF THE INVENTION

This invention provides novel, biological molecules for identification, detection, and/or regulation of cellular molecules involved in HIV pathogenesis. Specifically, in one aspect of the invention, it has been discovered that the expression of certain previously uncharacterized cellular genes is related to inhibition of apoptosis in HIV-1 infected cells. Accordingly, the apoptosis modulators of the invention may be used to advantage in the development of therapeutic agents for the treatment of HIV infection and AIDS.

In a preferred embodiment of the invention, isolated nucleic acid molecules are provided which encode human apoptosis modulating proteins, DA2 (also referred to interchangeably herein as HIV-Associated Life Preserver (HALP) or DNA-damage-inducible transcript 4 (DDIT4)), CD4, DF2, DF4, CC8, and DG1. An exemplary HALP nucleic acid molecule of the invention comprises the sequence of SEQ ID NO: 1. The human CD4 nucleic acid has the sequence of SEQ ID NO: 15 (GenBank accession number AL049356). The human DF2 nucleic acid has the sequence of SEQ ID NO: 14 (Genbank accession number AF164679). The human DF4 has the nucleic acid sequence of SEQ ID NO: 13 (GenBank accession number BC039361). The human CC8 nucleic acid has the sequence of SEQ ID NO: 12. The human DG1 nucleic acid has the sequence of SEQ ID NO: 16.

According to another aspect of the present invention, an isolated nucleic acid molecule is provided, which has a sequence selected from the group consisting of: (1) SEQ ID NO: 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16; (2) a sequence specifically hybridizing with preselected portions or all of the complementary strand of SEQ ID NO: 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16; (3) a sequence comprising preselected portions of SEQ ID NO: 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 (4) a complement of SEQ ID NO: 1, and (5) a sequence encoding part or all of a polypeptide encoded by the above-identified nucleic acid sequences. Such partial sequences are useful as probes to identify and isolate homologues of the apoptosis modulating nucleic acids of the invention. Accordingly, isolated nucleic acid sequences encoding natural allelic variants of the nucleic acids of SEQ ID NO: 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 are also contemplated to be within the scope of the present invention.

Host cells comprising the apoptosis modulator-encoding nucleic acid molecules of the invention are also contemplated to be within the scope of the present invention. Such host cells include, but are not limited to: bacterial cells, fungal cells, yeast cells, plant cells, insect cells, human cells and other animal cells. The HALP-encoding nucleic acid molecules may be conveniently cloned into a plasmid or retroviral vector for introduction into host cells. Such cells are useful in screening methods to identify compounds which modulate HALP expression. Compounds so identified may have therapeutic value in the treatment of patients infected with HIV.

According to another aspect of the present invention, isolated human apoptosis modulating proteins are provided. The loss of expression of these proteins is correlated with increased or decreased apoptosis of HIV-1 infected cells. FIGS. 15A and B (see Original Patent) set forth a series of pro-apoptotic and anti-apoptotic modulators, respectively, which can be employed in the screening methods described hereinbelow.

In another embodiment, the nucleic acid molecules of the invention may be used as diagnostic hybridization probes or as primers for diagnostic PCR analysis for identifying nucleic acids encoding apoptosis modulating proteins or mutations thereof. Antisense molecules are also provided herein and may be useful in the regulation of apoptosis modulator expression. Other methods encompassed by the present invention include immunodetection methods for assessing biological samples for the presence of apoptosis modulating protein.

According to another aspect of the invention, methods are provided for identifying agents which modulate (e.g., inhibit or activate) the activity of the apoptosis modulators described herein. The following summarizes methods employing the HALP encoding nucleic acid or protein, however any of the other apoptosis modulators of the invention, CC8, DF4, DF2, CD4 and DG1 may be employed in the following methods. An exemplary method comprises contacting cells expressing HALP, for example, with an agent suspected of having HALP-modulating activity under conditions whereby such an agent enters cells, and comparing apoptosis levels in cells in the presence and absence of the agent. Agents suspected of having HALP-modulating activity include, but are not limited to: an expression construct comprising HALP-encoding nucleic acid in an antisense orientation, an antisense HALP oligonucleotide, siRNA molecules engineered from the HALP sequence, antibodies immunologically specific for HALP which inhibit HALP function upon binding, and a variety of drugs and/or compounds. An increase in apoptosis in the presence of the agent is indicative of the agent's ability to inhibit HALP activity. A decrease in apoptosis in the presence of the agent is indicative of the agent's ability to activate HALP activity.

The screening methods described above may be practiced in HIV infected cells to identify those agents having antiviral activity. Antiviral activity can be assessed via termination or alteration in HIV particle or protein production levels in the presence and absence of such agents. Additionally, apoptosis in HIV infected cells can be measured in the presence and absence of such putative antiviral compounds.

The present invention also encompasses methods and compositions for the prevention and/or treatment of patients having diseases or conditions associated with aberrant apoptosis.

In yet another embodiment of the invention, small, interfering RNA (siRNA) molecules are provided which inhibit expression of HALP. Exemplary siRNA molecules have the sequences of SEQ ID NO: 21 and SEQ ID NO: 22.

Additionally, methods are provided for controlling expression of HALP in cells or tissues, e.g., human cells or tissues. The method comprises contacting cells or tissues with at least one siRNA molecule so that expression of HALP is altered. Alternatively, the at least one siRNA molecules may be encoded for in a vector which is brought into contact with the cells or tissue. Additionally, the siRNA molecules may be co-administered with at least one anti-HIV agent and/or at least one HIV vaccine.

In yet another embodiment of the invention, a pharmaceutical preparation is provided for treating a patient infected with HIV. The pharmaceutical preparation comprises at least one siRNA targeting HALP or at least one expression vector encoding for an siRNA targeting HALP. Such pharmaceutical preparations can be administered to a patient infected with HIV for treating the patient by eliminating or at least reducing the number of cells infected with HIV in the patient. The pharmaceutical preparations may additionally comprise at least one anti-HIV agent and/or at least one HIV vaccine.

DETAILED DESCRIPTION OF THE INVENTION

Ground-breaking research in basic biology has led to an understanding of some of the pathogenic mechanisms of HIV-1 infection. Analysis of T cells from individuals infected with HIV-1, or of T cells infected in vitro with HIV-1, demonstrated that a significant fraction of both infected and uninfected cells undergo apoptosis. The mechanism(s) whereby HIV-1 infected cells undergo apoptosis, however, remain obscure. Additional research using lymphoid tissues from HIV-1 infected humans and SIV-1 infected macaques has suggested that most infected cells are not apoptotic, whereas uninfected bystander T cells are apoptotic (3). One possible explanation for this observation is that HIV-1 infection confers a selective advantage to infected cells, thus rendering such cells resistant to or protected from apoptosis. In addition, HIV-1 and host cellular targets may both be actively involved in the regulation of apoptosis during HIV-1 infection. Hence, the identification of viral and host cell-specific targets that modulate apoptosis in cells infected with HIV-1 is essential for elucidation of the mechanism(s) by which HIV-1 kills T cells. Such knowledge may be used to advantage to identify and provide agents (e.g., drugs, compounds) that induce apoptotic pathways in HIV-1 infected cells in patients.

To this end, a PCR-based subtractive hybridization method was applied to identify genes that play a role in modulating apoptosis in HIV-1 infected cells (Examples I and II of the present invention). Subtractive cloning is a powerful technique that facilitates isolation and cloning of mRNA transcripts which are differentially expressed in different cell populations. In general, a subtraction scheme involves a tracer (+) cell population and a driver (-) cell population and provides means to identify mRNA transcripts expressed in the tracer and not in the driver cell population. Briefly, nucleic acid (cDNA or mRNA) from a tracer cell population is allowed to hybridize with an excess of nucleic acid from a driver cell population to ensure that a high percentage of the tracer nucleic acid forms hybrids. Tracer/driver hybrids comprise nucleic acid sequences common to both cell populations. Hybrids comprised of complementary tracer and driver nucleic acid, and all the driver nucleic acid, are subsequently removed in a subtraction step. The remaining unhybridized fraction is enriched for those nucleic acid sequences expressed preferentially in the tracer cell population. Such methods are well known to those of skill in the art and have been described in detail elsewhere (e.g., Unit 5.9 of Ausubel et al. (27)).

As described herein and known in the art, reciprocal subtractions may be performed. Reciprocal subtractive hybridizations provide means to identify and isolate genes which are preferentially expressed in either of the different cell populations. Reciprocal subtractions may be used to isolate genes preferentially expressed in cell population A (used as a tracer) compared to B (used as a driver) and genes preferentially expressed in cell population B (used as a tracer) compared to A (used as a driver). Such methods utilize PCR to amplify cDNA after each subtraction to prepare sufficient quantities of tracer and driver nucleic acids for the next subtraction. The progress of the subtraction process may be monitored by a variety of means, including slot blot hybridization to detect residual levels of transcripts encoding housekeeping genes. Differentially expressed cDNA sequences may be used to construct a subtracted cDNA library.

As described herein, a reciprocal subtractive hybridization was performed to identify genes preferentially expressed in HIV-1 infected cell populations undergoing apoptotis and to identify genes preferentially expressed in HIV-1 infected cell populations that are viable and stable (non-apoptotic). Genes preferentially expressed during apoptotic stages of HIV-1 infection are designated as having pro-apoptotic activity, whereas genes preferentially expressed during non-apoptotic stages of HIV-1 infection are designated as possessing anti-apoptotic activity. Additionally, a series of known pro-apoptotic and anti-apoptotic genes are provided in FIGS. 15A and B.

Thus, in accordance with the present invention, six novel host cell nucleic acids, designated HALP, CC8, DF4, DF2, CD4 and DG1 have been identified which regulate apoptosis in HIV-1 infected T cells. All of these nucleic acids and their encoded proteins are referred to herein collectively as apoptosis modulators and thus maybe utilized in the screening methods of the invention. Specifically, increases in apoptosis modulator levels are correlated with down-regulation or inhibition of apoptotic pathways in HIV-1 infected cells. Based on their expression pattern during the course of viral infection, therefore, the apoptosis modulators of the invention have been designated as having anti-apoptotic activity. Such apoptosis modulator mediated activity may have critical consequences in the development of viral latency. The apoptosis modulating compositions of the invention may be used to advantage to: (1) identify additional cellular targets that regulate HIV-1 infection; and (2) facilitate the development of novel therapeutic agents which inhibit the cellular activity of apoptosis modulators thereby promoting apoptosis of HIV-1 infected cells and preventing HIV-1 latency. Agents so developed may be used in combination with standard anti-retroviral therapy to ameliorate HIV-1 infection.

While HIV-1 is exemplified herein, the present invention encompasses method for screening agents having efficacy against other retroviruses, including without limitation HIV-2, simian immunodeficiency virus (SIV), and feline immunodeficiency virus (FIV).
II. Preparation of Apoptosis Modulator Encoding Nucleic Acid Molecules:

In accordance with the present invention, six cellular genes have been identified with are involved in regulation of apoptosis in HIV infected cells. While the following exemplifies preparation of HALP encoding nucleic acids, any of the other nucleic acid molecules encoding the apoptosis modulators described herein, CC8, DF2, DF4, DG1, and CD4 may be prepared in a comparable manner.

A full length nucleic acid sequence encoding HALP (SEQ ID NO: 1; 714 bp) is shown in FIG. 6 (see Original Patent). The full length cDNA was generated based on reverse transcription polymerase chain reaction (RT-PCR) utilizing HALP-specific primers CTG TCC TCA CCA TGC CTA (SEQ ID NO: 10; 5' primer) and TGA AGT TCA ACA CTC CTC AA (SEQ ID NO: 11; 3' primer) whose sequences were based on those of a partial HALP nucleic acid (SEQ ID NO: 9; 415 bp; FIG. 7 (see Original Patent)) isolated by subtractive hybridization. All sequences were determined using ABI PRISM.RTM. BigDye.TM. Primer Cycle Sequencing Kits at the Nucleic Acid/Protein Core in The Children's Hospital of Philadelphia utilizing routine methodology.

Nucleic acid molecules of the invention encoding apoptosis modulator polypeptides may be prepared by two general methods: (1) synthesis from appropriate nucleotide triphosphates, or (2) isolation from biological sources. Both methods utilize protocols well known in the art. The availability of nucleotide sequence information, such as the DNA sequences encoding HALP, enables preparation of an isolated nucleic acid molecule of the invention by oligonucleotide synthesis. Synthetic oligonucleotides may be prepared by the phosphoramidite method employed in the Applied Biosystems 38A DNA Synthesizer or similar devices. The resultant construct may be used directly or purified according to methods known in the art, such as high performance liquid chromatography (HPLC).

Specific probes for identifying such sequences as the apoptosis modulator encoding sequence may be between 15 and 40 nucleotides in length. For probes longer than those described above, the additional contiguous nucleotides are provided within the corresponding SEQ ID NO.

Additionally, cDNA or genomic clones having homology with apoptosis modulators may be isolated from other species, including without limitation feline and simian species, using oligonucleotide probes corresponding to predetermined sequences within the apoptosis modulator nucleic acids of the invention. Such homologous sequences encoding apoptosis modulators may be identified by using hybridization and washing conditions of appropriate stringency. For example, hybridizations may be performed, according to the method of Sambrook et al. (28) using a hybridization solution comprising: 5.times.SSC, 5.times. Denhardt's reagent, 1.0% SDS, 100 .mu.g/ml denatured, fragmented salmon sperm DNA, 0.05% sodium pyrophosphate and up to 50% formamide. Hybridization is carried out at 37-42.degree. C. for at least six hours. Following hybridization, filters are washed as follows: (1) 5 minutes at room temperature in 2.times.SSC and 1% SDS; (2) 15 minutes at room temperature in 2.times.SSC and 0.1% SDS; (3) 30 minutes-1 hour at 37.degree. C. in 1.times.SSC and 1% SDS; (4) 2 hours at 42-65.degree. C. in 1.times.SSC and 1% SDS, changing the solution every 30 minutes.

One common formula for calculating the stringency conditions required to achieve hybridization between nucleic acid molecules of a specified sequence homology (28) is as follows: T.sub.m=81.5.degree. C.+16.6 Log [Na+]+0.41 (% G+C)-0.63 (% formamide)-600/#bp in duplex

As an illustration of the above formula, using [Na+]=[0.368] and 50% formamide, with GC content of 42% and an average probe size of 200 bases, the T.sub.m is 57.degree. C. The T.sub.m of a DNA duplex decreases by 1-1.5.degree. C. with every 1% decrease in homology. Thus, targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42.degree. C.

The stringency of the hybridization and wash depend primarily on the salt concentration and temperature of the solutions. In general, to maximize the rate of annealing of the probe with its target, the hybridization is usually carried out at salt and temperature conditions that are 20-25.degree. C. below the calculated T.sub.m of the hybrid. Wash conditions should be as stringent as possible for the degree of identity of the probe for the target. In general, wash conditions are selected to be approximately 12-20.degree. C. below the T.sub.m of the hybrid. In regards to the nucleic acids of the current invention, a moderate stringency hybridization is defined as hybridization in 6.times.SSC, 5.times. Denhardt's solution, 0.5% SDS and 100 .mu.g/ml denatured salmon sperm DNA at 42.degree. C., and washed in 2.times.SSC and 0.5% SDS at 55.degree. C. for 15 minutes. A high stringency hybridization is defined as hybridization in 6.times.SSC, 5.times. Denhardt's solution, 0.5% SDS and 100 .mu.g/ml denatured salmon sperm DNA at 42.degree. C., and washed in 1.times.SSC and 0.5% SDS at 65.degree. C. for 15 minutes. A very high stringency hybridization is defined as hybridization in 6.times.SSC, 5.times. Denhardt's solution, 0.5% SDS and 100 .mu.g/ml denatured salmon sperm DNA at 42.degree. C., and washed in 0.1.times.SSC and 0.5% SDS at 65.degree. C. for 15 minutes.

The nucleic acid molecules described herein include cDNA, genomic DNA, RNA, and fragments thereof which may be single- or double-stranded. Thus, nucleic acids are provided having sequences capable of hybridizing with at least one sequence of a nucleic acid sequence, such as selected segments of a sequence encoding an apoptosis modulator. Also contemplated in the scope of the present invention are methods of use for oligonucleotide probes which specifically hybridize with the DNA from the sequences encoding the apoptosis modulators under high stringency conditions. Primers capable of specifically amplifying the sequences encoding apoptosis modulators are also provided. As mentioned previously, such oligonucleotides are useful as primers for detecting, isolating and amplifying sequences encoding apoptosis modulators.

Antisense nucleic acid molecules which may be targeted to translation initiation sites and/or splice sites to inhibit the expression of the apoptosis modulator genes or production of their encoded proteins are also provided. Such antisense molecules are typically between 15 and 30 nucleotides in length and often span the translational start site of mRNA molecules. Antisense constructs may also be generated which contain the entire apoptosis modulator sequence in reverse orientation.

Small interfering RNA (siRNA) molecules designed to inhibit expression of the apoptosis modulator genes are also provided. Typically, siRNA molecules are double-stranded RNA molecules between about 12 and 30 nucleotides in length, more typically about 21 nucleotides in length. The nucleotide sequence of the siRNA molecules commonly begin from an AA dinucleotide sequence near the AUG start codon but not within about 75 bases of said start codon. The siRNA molecules typically have a GC content of between about 45% and about 55% and ideally do not contain stretches of more than 3 guanosine bases in a row.

III. Preparation of Apoptosis Modulating Proteins and Antibodies Immunologically Specific Therefore:

The apoptosis modulators of the present invention may be prepared in a variety of ways, according to known methods. The proteins may be purified from appropriate sources, e.g., human cells or tissues as described in detail in Example 1.

The identification of nucleic acid molecules encoding apoptosis modulators facilitates the expression of apoptosis modulators in vitro by methods known in the art. For example, a cDNA or gene may be cloned into an appropriate in vitro transcription vector, such as a pSP64 or pSP65 vector for in vitro transcription, followed by cell-free translation in a suitable cell-free translation system, such as wheat germ or rabbit reticulocytes. In vitro transcription and translation systems are commercially available, e.g., from Promega Biotech, Madison, Wis. or BRL, Rockville, Md.

According to a preferred embodiment, larger quantities of apoptosis modulators may be produced by expression in a suitable procaryotic or eucaryotic system. For example, part or all of a DNA molecule may be inserted into a plasmid vector adapted for expression in a bacterial cell (such as E. coli) or a yeast cell (such as Saccharomyces cerevisiae), or into a baculovirus vector for expression in an insect cell. Such vectors comprise the regulatory elements necessary for-expression of the DNA in the host cell, positioned in such a manner as to permit expression of the DNA in the host cell. Such regulatory elements required for expression include promoter sequences, translation control sequences and, optionally, enhancer sequences.

The apoptosis modulator protein produced by gene expression in a recombinant procaryotic or eucaryotic system may be purified according to methods known in the art. In a preferred embodiment, the recombinant protein contains several (e.g., 6-8) histidine residues on the amino or carboxyl termini, which allows the protein to be affinity purified on a nickel column. If histidine tag-vectors are not used, an alternative approach involves purifying the recombinant protein by affinity separation, such as by immunological interaction with antibodies that bind specifically to the recombinant protein. Such methods are commonly used by skilled practitioners.

Apoptosis modulator protein, prepared by the aforementioned methods, may be analyzed according to standard procedures. Methods for analyzing the HALP-inhibitory activity of apoptosis are set forth in U.S. Pat. Nos. 5,976,786 and 6,046,007, the disclosures of which are incorporated by reference herein.

The present invention also provides antibodies capable of immunospecifically binding to proteins of the invention. Polyclonal or monoclonal antibodies immunologically specific for epitopes of apoptosis modulators may be prepared according to standard methods. Monoclonal antibodies may be prepared according to general methods of Kohler and Milstein, following standard protocols. Polyclonal and/or monoclonal antibodies may be prepared as described in several laboratory protocol handbooks, including: Sambrook et al. (28); Ausubel et al. (27), and Harlow and Lane (29). In a preferred embodiment, antibodies are prepared, which react immunospecifically with various epitopes of the apoptosis modulators of the invention.

Polyclonal or monoclonal antibodies that immunospecifically interact with the apoptosis modulators may be utilized for identifying and purifying such proteins. For example, antibodies may be utilized for affinity separation of proteins with which they immunospecifically interact. Antibodies may also be used to immunoprecipitate proteins from a sample containing a mixture of proteins and other biological molecules. Antibodies may also be used to bind an apoptosis modulator molecule, thereby rendering the apoptosis modulator inactive and/or unstable. Antibodies capable of binding to apoptosis modulators and inhibiting apoptosis modulator function are known as inhibitory antibodies. Such apoptosis modulator inhibitory antibodies may be used to advantage to induce apoptosis in HIV-1 infected cells in vitro and in vivo. Such apoptosis modulator inhibitory antibodies are of particular utility for the therapeutic treatment of HIV-1 patients to facilitate induction of apoptosis in HIV-1 infected cells, thus eradicating such cells from the patient.

IV. Uses of Apoptosis Modulators Encoding Nucleic Acid Molecules, Proteins and Antibodies:

A. Nucleic Acids Encoding Apoptosis Modulators

Nucleic acids encoding apoptosis modulator protein may be used for a variety of purposes in accordance with the present invention. DNA, RNA, or fragments thereof encoding apoptosis modulators may be used as probes to detect the presence of and/or expression of such genes. Methods in which nucleic acids encoding apoptosis modulators may be utilized as probes for such assays include, but are not limited to: (1) in situ hybridization; (2) Southern hybridization (3) Northern hybridization; and (4) assorted amplification reactions such as polymerase chain reactions (PCR).

The nucleic acids of the invention may also be utilized as probes to identify related genes from other animals and microbes. As is well known in the art, hybridization stringencies may be adjusted to allow hybridization of nucleic acid probes with complementary sequences of varying degrees of homology.

Nucleic acid molecules, or fragments thereof, encoding apoptosis modulators may also be utilized to control the production of apoptosis modulators, thereby regulating the amount of protein available to participate in the inhibition of apoptosis in HIV-1 infected cells. As mentioned above, antisense oligonucleotides corresponding to essential processing sites in apoptosis modulator mRNA molecules, siRNA molecules, or other gene silencing approaches may be utilized to inhibit apoptosis modulator production in targeted cells, such as T cells. Alterations in the physiological amount of apoptosis modulators may dramatically affect the activity of other protein factors involved in the induction or maintenance of retroviral infection including HIV-1, HIV-2, FIV, and SIV infections.

The nucleic acid molecules of the invention may also be used to advantage to identify mutations in apoptosis modulator encoding nucleic acids from HIV-1 infected cells. Nucleic acids may be isolated from HIV-1 infected patients and contacted with the sequences of the invention under conditions where hybridization occurs between sequences of sufficient complementarity. Such duplexes may then be assessed for the presence of mismatched DNA. Mismatches may be due to the presence of a point mutation, insertion or deletion of nucleotide molecules. Detection of such mismatches may be performed using methods well known to those of skill in the art.

Nucleic acids encoding apoptosis modulators may also be introduced into host cells. In a preferred embodiment, HIV-1 infected T lymphoblastoid cells are provided which comprise an apoptosis modulator protein encoding nucleic acid such as SEQ ID NO: 1 or a variant thereof. Host cells contemplated for use include, but are not limited to, human, bacteria, yeast, insect and other animal cells. The nucleic acids may be operably linked to appropriate regulatory expression elements suitable for the particular host cell to be utilized. Methods for introducing nucleic acids into host cells are well known in the art. Such methods include, but are not limited to, transfection, transformation, calcium phosphate precipitation, electroporation and lipofection.

Host cells or extracts prepared therefrom containing apoptosis modulators may be used as screening tools to identify compounds which modulate their activity. Test compounds may be evaluated for their ability to inhibit HALP activity, for example, as assayed by activation of apoptotic pathways in and/or apoptosis of HIV-1 infected cells. Changes in apoptotic pathways may be measured using a variety of techniques, including, but not limited to those described in Example I. Modulation of apoptosis modulator activity in the context of a host cell may also be detected by changes in the level of apoptosis modulator gene expression (e.g., RNA or protein). Modulation of HALP activity, for example, may be assessed by measuring alterations in HALP binding activity or HALP biological activity in the presence of a test compound. Test compounds may also be assessed for the induction and/or suppression of expression of genes regulated by HALP.

The host cells described above may also be "primed" or "induced" to undergo apoptosis. Host cells induced to undergo apoptosis and extracts prepared thereof containing apoptosis modulators may be used as screening tools to identify compounds which alter protein activity. In vitro methods for inducing apoptosis are well known in the art and may comprise providing cells or tissues having cell surface receptors capable of mediating apoptosis such as a T cell receptor (TCR), a tumor necrosis factor (TNF) receptor, or a Fas receptor. Such methods comprise culturing cells under conditions (temperature, growth or culture medium and gas (CO.sub.2)) and for an appropriate amount of time to attain exponential proliferation without density dependent constraints. Populations of cells undergoing exponential growth may be exposed to preliminary conditions necessary for apoptosis, an effective amount of an inducing agent, e.g., a TCR ligand, TNF, or a Fas ligand such as an anti-Fas antibody may be added to the culture. Anti-Fas antibodies and mitogens (ConA) capable of inducing apoptosis are well known to those of skill in the art (18, 19). Such cells are now "induced" to undergo apoptosis and may be cultured under suitable temperature and time conditions.

In one embodiment, cells are transfected with an effective amount of nucleic acid encoding an apoptosis modulators protein or a fragment thereof and the cells are cultured under suitable temperature and time conditions to inhibit apoptosis. The apoptosis modulator encoding nucleic acid may be transfected prior to, simultaneously with, or after, an apoptosis inducing agent. The cells are assayed for apoptotic activity using methods well known to those of skill in the art and described herein. In other embodiments, a drug or compound to be tested for the ability to alter apoptosis modulator activity may be added in varying concentrations at a time that is simultaneous with, prior to, or after the inducing agent. Those of skill in the art would recognize that such analyses must be performed with appropriate experimental controls.

Thus, the compositions and methods of the present invention provide a powerful in vitro drug screening bioassay of utility in the identification of drugs that are agonists or antagonists of apoptosis modulator function in host cells. Thus, one can screen for drugs capable of antagonizing or inhibiting apoptosis modulator activity that promote cellular apoptosis. One may also screen for drugs having similar or enhanced ability to prevent or inhibit apoptosis. One of skill in the art may determine when a drug is, for example, capable of enhancing HALP-mediated inhibition of apoptosis by noting a decrease in the number of morphological changes associated with apoptosis or a reduction in cell death. The in vitro method further provides an assay to determine if the methods and compositions of the invention are useful to treat a patient with a pathological condition or disease that has been linked to aberrant apoptotic cell death.

The availability of apoptosis modulator-encoding nucleic acids also enables the production of HIV-1 infected cells carrying part or all of an apoptosis modulator gene or mutated sequences thereof, in single or amplified copies.

The alterations to the apoptosis modulator genes envisioned herein include modifications, deletions, and substitutions. Such modifications, deletions or substitutions can result in an apoptosis modulator protein having altered characteristics or functions. As used herein, a "targeted gene" or "knock-out" is a DNA sequence introduced into host cells by way of human intervention, including but not limited to, the methods described herein. The targeted genes of the invention include DNA sequences which are designed to specifically alter apoptosis modulator regulation (e.g., inhibition or activation) of apoptosis in HIV-1 infected cells.

As described above, the apoptosis modulator-encoding nucleic acids are also used to advantage to produce large quantities of substantially pure proteins, or selected portions thereof.

B. Apoptosis Modulator Protein and Antibodies

Purified apoptosis modulator protein, or fragments thereof, may be used to produce polyclonal or monoclonal antibodies which also may serve as sensitive detection reagents for the presence and accumulation of HALP, for example, (or complexes containing HALP) in blood samples or cultured cells. Recombinant techniques enable expression of fusion proteins containing part or all of the apoptosis modulators of the invention. The full length protein or fragments of the protein may be used to advantage to generate an array of monoclonal antibodies specific for various epitopes of the protein, thereby providing even greater sensitivity for detection of the protein in HIV-1 infected cells.

Polyclonal or monoclonal antibodies immunologically specific for HALP, for example, may be used in a variety of assays designed to detect and quantitate the protein. Such assays include, but are not limited to: (1) flow cytometric analysis; (2) immunochemical localization of HALP in HIV-1 infected cells; and (3) immunoblot analysis (e.g., dot blot, Western blot) of extracts from leukocytes. Additionally, as described above, anti-HALP antibodies may be used for purification of HALP (e.g., affinity column purification, immunoprecipitation).

Antibodies immunologically specific for apoptosis modulators may also be used to inhibit the activity of apoptosis modulators in virus-infected (e.g., HIV-1) cells. Targeted delivery of anti-apoptosis modulator inhibitory antibodies to HIV-1 infected cells may be achieved by utilizing anti-gp120-antibody-studded liposomes comprising, for example, sfv-encoding antibodies immunologically specific for an apoptosis modulator. Methods for the production of such anti-gp120-antibody-studded liposomes and administration of targeted liposomes to patients have been previously described in Maulik et al. (20) and U.S. Pat. No. 5,709,879 (Barchfeld et al.); U.S. Pat. No. 5,935,937 (Smith); U.S. Pat. No. 5,981,279 (Weiss); and U.S. Pat. No. 6,025,193 (Weiss), the entire contents of which are incorporated herein by reference. In one aspect, sfv-encoding apoptosis modulator antibodies may be used which are specific for epitopes identified as essential for apoptosis modulator function and/or activity (e.g., anti-apoptotic activity). The binding of such sfv antibodies to HALP, for example, in HIV-1 infected cells mediates inhibition of HALP anti-apoptotic activity and thus, induces apoptosis of infected cells.

According to another aspect of the invention, methods of screening drugs for the treatment of HIV-1 infection which inhibit apoptosis modulator activity are provided. Inhibition of apoptosis modulator activity by gene transfer or by pharmacological means (e.g., small molecules which bind apoptosis modulators and thereby alter apoptosis modulator structure and/or function) would be expected to promote apoptotic activity of HIV-1 infected host cells.

The apoptosis modulator polypeptide or fragment employed in drug screening assays may either be free in solution, affixed to a solid support or within a cell. One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant polynucleotides expressing the polypeptide or fragment, preferably in competitive binding assays. Upon expression of an apoptosis modulator polypeptide or fragment thereof, cells may be disrupted (e.g., by detergent or mechanical lysis) to produce a cellular lysate which may be affixed to a solid support for use in competitive binding assays. Such cells, either in viable or fixed form, may also be used for standard binding assays. One may determine, for example, formation of complexes between an apoptosis modulator polypeptide or fragment and the agent being tested, or examine the degree to which the formation of a complex between a apoptosis modulator polypeptide or fragment and a known ligand is interfered with by the agent being tested.

Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to an apoptosis modulator and is described in detail in Geysen, PCT published application WO 84/03564, published on Sep. 13, 1984. Briefly stated, large numbers of different, small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with an apoptosis modulator polypeptide and washed. Bound apoptosis modulator polypeptide is then detected by methods well known in the art.

Purified apoptosis modulators can be coated directly onto plates for use in the aforementioned drug screening techniques. However, non-neutralizing antibodies to the polypeptides can be used to capture antibodies to immobilize the apoptosis modulator polypeptide on the solid phase.

The present invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of specifically binding the apoptosis modulator polypeptide compete with a test compound for binding to the apoptosis modulator polypeptide or fragments thereof. In this manner, the antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants of the apoptosis modulator polypeptide.

The goal of rational drug design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact (e.g., agonists, antagonists, inhibitors) in order to fashion drugs which are, for example, more active or stable forms of the polypeptide, or which, e.g., enhance or interfere with the function of a polypeptide in vivo. See, e.g., Hodgson (21). In one approach, one first determines the three-dimensional structure of a protein of interest (e.g., apoptosis modulator) or, for example, of an apoptosis modulator containing complex, by x-ray crystallography, by nuclear magnetic resonance, by computer modeling or most typically, by a combination of approaches. Less often, useful information regarding the structure of a polypeptide may be gained by modeling based on the structure of homologous proteins. An example of rational drug design is the development of HIV protease inhibitors (22). In addition, peptides (e.g., HALP polypeptide) may be analyzed by an alanine scan (23). In this technique, an amino acid residue is replaced by Ala, and its effect on the peptide's activity is determined. Each of the amino acid residues of the peptide is analyzed in this manner to determine the important regions of the peptide.

It is also possible to isolate a target-specific antibody, selected by a functional assay, and then to solve its crystal structure. In principle, this approach yields a pharmacore upon which subsequent drug design can be based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies (anti-ids) to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of the anti-ids would be expected to be an analog of the original molecule. The anti-id could then be used to identify and isolate peptides from banks of chemically or biologically produced banks of peptides. Selected peptides would then act as the pharmacore.

Thus, one may design drugs which have, e.g., improved apoptosis modulator activity or stability or which act as inhibitors, agonists, antagonists, etc. of such activity. By virtue of the availability of cloned apoptosis modulator sequences, sufficient amounts of the polypeptide may be made available to perform such analytical studies as x-ray crystallography. In addition, the knowledge of the protein sequences provided herein will guide those employing computer modeling techniques in place of, or in addition to x-ray crystallography.

V. Compositions Comprising Apoptosis Modulator Nucleic Acid Molecules, Proteins and Antibodies and Methods of Use Thereof:

Nucleic acid encoding apoptosis modulators, in either sense or antisense orientation, apoptosis modulator polypeptides and fragments thereof and antibodies thereto, and drugs or compounds identified by the methods of the present invention as having apoptosis modulating activity may be used to advantage as agents for the therapeutic treatment of an individual with a disease or condition which is linked to aberrant cellular apoptosis. Agents of the present invention may be administered using therapeutically effective administration protocols. Such protocols comprise suitable dose parameters and modes of administration that result in alleviation of symptoms related to a disease or condition which is linked to aberrant cellular apoptosis.

As used herein, the term "administering" for in vivo and ex vivo purposes refers to providing a subject with an amount of a nucleic acid molecule, polypeptide or antibody thereto sufficient to modulate apoptosis of a target cell. Methods of administering pharmaceutical compositions are well known to those of skill in the art and include, but are not limited to, microinjection, topical, oral, local, intravenous, subcutaneous, intramuscular, and parenteral administration. Administration may be effected continuously or intermittently throughout the course of treatment. Methods for determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the vector used for therapy, the nucleic acid used for therapy, the polypeptide used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations may be carried out with the dose level and pattern being selected by the attending physician. For example, the compositions may be administered to a subject prior to or after onset of a disease or condition that is associated with aberrant cellular apoptosis.

As used herein, a "therapeutically effective amount" of an agent or composition of the present invention is an amount sufficient to modulate apoptotic pathways in a patient with a disease or condition that is associated with aberrant cellular apoptosis.

In one embodiment, an expression construct comprising HALP-encoding nucleic acid in an antisense orientation, antisense HALP oligonucleotides, HALP siRNA molecules, or inhibitory antibodies immunologically specific for HALP for example, may be used to advantage as therapeutic agents to limit infection by an immunodeficiency virus (e.g., HIV-1) and/or prevent or treat AIDS in a patient. Therapeutically effective dose parameters and modes of administration may be determined using methods standard in the art. Such methods include, for example, determination of survival rates, side effects (i.e., toxicity) and progression or regression of disease. For example, a therapeutically effective dose and mode of administration for a formulation of the present invention may be determined by assessing response rates. Such response rates refer to the percentage of treated patients that responds with either partial or complete remission.

For the treatment of HIV-1 related diseases, a therapeutically effective dose of an agent or composition administered according to the present invention may depend on the viral load or cell tropism, as well as differences in levels of expression of immunodeficiency virus genes encoding apoptosis regulator proteins [e.g., HALP (disclosed herein), Vpr, Tat, Vif, Nef, Gag, and Vpu].

It will be obvious to one of skill in the art that the number of doses administered to a patient infected with an immunodeficiency virus is dependent upon the extent of the infection and the response of an individual to the treatment. For example, a patient with a high titer of HIV may require more doses than a patient with a lower titer. In some cases, however, a patient with a high titer of HIV may require fewer doses than a patient with a lower titer, if the former patient responds more favorably to the therapeutic composition than the latter patient. Thus, it is within the scope of the present invention that a suitable number of doses, as well as the time periods between administration, includes any number required to cause regression of a disease.

A therapeutic composition comprising agents of the present invention may also comprise a pharmaceutically acceptable carrier. As used herein, a "pharmaceutically acceptable carrier" refers to any substance suitable as a vehicle for delivering an agent of the present invention to a suitable in vitro or in vivo site of action. As such, carriers can act as a pharmaceutically acceptable excipient or formulation of a therapeutic composition containing agents. Preferred carriers are capable of maintaining agents of the present invention in a form that is capable of modulating (e.g., enhancing or inhibiting progression) of apoptosis of a cell. Examples of such carriers include, but are not limited to water, phosphate buffered saline, Ringer's solution, dextrose solution, serum-containing solutions, Hank's solution and other aqueous physiologically balanced solutions. Aqueous carriers may also contain suitable auxiliary substances required to approximate the physiological conditions of the recipient, for example, by enhancing chemical stability and isotonicity. Suitable auxiliary substances include, for example, sodium acetate, sodium chloride, sodium lactate, potassium chloride, calcium chloride, and other substances used to produce phosphate buffer, Tris buffer, and bicarbonate buffer. Auxiliary substances may also include preservatives, such as thimerosal, m- and o-cresol, formalin and benzol alcohol. Preferred auxiliary substances for aerosol delivery include surfactant substances non-toxic to a recipient, for example, esters or partial esters of fatty acids containing from about six to about twenty-two carbon atoms. Examples of esters include, caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric, and oleic acids. Formulations of the present invention may be sterilized by conventional methods and/or lyophilized.

Useful carriers for agents of the present invention include any artificial or natural lipid-containing target molecule, preferably cells, cellular membranes, liposomes, and micelles. Preferably, formulations of the present invention are administered in the form of liposomes or micelles. Liposomes and micelles of the present invention are capable of delivering an agent from the extracellular space of a cell to the intracellular space of a cell. Agents of the present invention are combined with liposomes or micelles to provide a means for the delivery of a therapeutically effective amount of an agent to a cell such that the progression of apoptosis in such a cell is altered. Lipid-based carriers are particularly useful for embodiments of the present invention wherein a therapeutic composition comprises a nucleic acid molecule or antibody. Such delivery systems are known and have been successfully applied in the art and are discussed in Maulik et al. (20) and U.S. Pat. No. 5,709,879 (Barchfeld et al.); U.S. Pat. No. 5,935,937 (Smith); U.S. Pat. No. 5,981,279 (Weiss); and U.S. Pat. No. 6,025,193 (Weiss) which are incorporated by reference herein in their entirety.

In another embodiment, a "therapeutically effective amount" of a composition of the present invention may be administered to a patient to inhibit or at least partially arrest apoptosis and the accompanying pathology, such as is observed in a variety of disorders characterized by inordinate cellular apoptosis. Such diseases include but are not limited to acute and chronic inflammatory disease, leukemia, myocardial infarction, stroke, traumatic brain injury, neural and muscular degenerative diseases, aging, tumor induced-cachexia, hair loss, rheumatoid arthritis, and systemic lupus erythematosus.

The compositions may also be administered to subjects or individuals susceptible to or at risk of developing apoptosis-related disease to prevent pathological cell death. In one embodiment, the composition may be administered to a subject susceptible to a neural degenerative disease to maintain neuronal cell function and viability. In such embodiments, a "prophylactically effective amount" of the composition may be administered to maintain normal cellular viability and function.

It should be understood that by preventing or inhibiting unwanted cell death in a subject or individual, the compositions and methods of the invention also provide methods for treating, preventing and/or ameliorating the symptoms associated with a disease characterized by inordinate apoptosis of cells. Thus, the compositions and methods of the invention provide means to maintain cellular viability in patients with diseases associated with excessive and/or inappropriate apoptotic cell death.

VI. siRNA Molecules

As discussed hereinabove, small, interfering RNA (siRNA) molecules are typically double stranded RNA molecules (RNA is usually single stranded) which inhibit expression of its target mRNA (see, e.g. reference 27, particularly chapter 26). As used herein, the term siRNA may include what is sometimes referred to as short hairpin RNA (shRNA) molecules. Typically, shRNA molecules consist of short complementary sequences separated by a small loop sequence wherein one of the sequences is complimentary to the gene target. shRNA molecules are typically processed into an siRNA within the cell by endonucleases.

Chemically modified siRNA molecules may be employed in the instant invention. Non-limiting examples of such chemical modifications include, without limitation, phosphorothioate internucleotide linkages, 2'-O-methyl ribonucleotides, 2'-deoxy-2'-fluoro ribonucleotides, 2'-deoxy ribonucleotides, "universal base" nucleotides, 5-C-methyl nucleotides, and inverted deoxyabasic residue incorporation. Preferably, the chemical modifications preserve the inhibition activity of the unmodified siRNA molecule in cells while, at the same time, increasing the serum stability of these compounds or other favorable property of the siRNA molecules. U.S. patent application Publication No. 20050032733, incorporated herein by reference, provides numerous suitable modifications of siRNA molecules.

Vectors for the expression of siRNA molecules preferably employ a strong promoter which may be constitutive or regulated. Such promoters are well known in the art and include, but are not limited to, RNA polymerase II promoters, the T7 RNA polymerase promoter, and the RNA polymerase III promoters U6 and H1 (see, e.g., Myslinski et al. (2001) Nucl. Acids Res., 29:2502-09). Preferably, RNA polymerase III promoters are employed. Preferable expression vectors for expressing the siRNA molecules of the invention are plasmids and viral vectors (see, e.g., Sui et al. (2002) PNAS 99:5515-5520; Xia et al. (2002) Nature Biotech. 20:1006-1010; Barton and Medzhitov (2002) PNAS 99:14943-14945; Brummelkamp et al. (2002) Science 296:550-553; Devroe and Silver (2002) BMC Biotechnol., 2(1):15; Tiscornia et al. (2003) PNAS, 100:1844-1848).

siRNA molecules targeting HALP may be administered alone or in combination with other siRNA molecules targeting other regions of HALP and/or at least one anti-HIV agents and/or at least one HIV vaccine. Anti-HIV agents include, without limitation: protease inhibitors (e.g., Indinavir, Ritonavir, Saqinavir, Nelfinavir, and Amprenavir), nucleoside reverse transcriptase inhibitors (e.g., Zidovudine (AZT), Didanosine, Zalcitabine, Lamivudine, Stavudine, and Abacavir), non-nucleoside reverse transcriptase inhibitors (e.g., Nevirapine, Delavirdine, and Efavirenz), integrase inhibitors, and fusion inhibitors.
 

Claim 1 of 12 Claims

1. An isolated small, interfering RNA (siRNA) molecule targeted to a nucleic acid molecule encoding HIV-Associated Life Preserver (HALP), wherein said siRNA molecule inhibits expression of HALP protein upon entry into a cell comprising HALP encoding nucleic acids, wherein the sequence of the sense strand of said siRNA molecule is SEQ ID NO: 21.

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