Patent Application
20130065819
The present invention relates to compositions and methods to treat or prevent viral infections. In particular, the invention relates to compositions and methods to treat or prevent HIV infections, as well as to modulate HIV replication.
According to figures released by the World Health Organization in December 2008 there are approximately 33 million people worldwide that are living with HIV infection, with 2.7 million newly reported infections and 2 million deaths yearly. Furthermore, the Public Health Agency of Canada reports a steady number of newly diagnosed cases over the past 12 years (HIV and AIDS in Canada: Surveillance Report to Dec. 31, 2008, Public Health Agency of Canada, 2009), with an estimated 58,000 Canadians living with HIV/AIDS. The scope of this global pandemic underscores the need for continued research efforts to find new avenues for prevention and treatment strategies.
While there is no current vaccine available to prevent HIV infection, for infected individuals there are a variety of anti-retroviral medications available that fall into four broad categories: Reverse transcriptase (RT) inhibitors, protease inhibitors, integrase inhibitors, viral entry inhibitors, and combinations thereof known as HAART (highly active antiretroviral therapy). While these therapies have undeniably improved the prognosis for HIV patients, there remain a number of drawbacks. These include a lack of complete viral suppression, as well as numerous, and sometimes serious, side effects (1). For these reasons intense research is focused on finding new avenues to combat the disease by developing drug-targeting strategies against key mediators of viral pathogenesis. For example, continued efforts are aimed at novel vaccine development strategies and recent attention has focused on the HIV Tat transcription factor as a drug target. This is due to its crucial role in a variety of viral functions including early RNA splicing, replication and transcription (2).
Upon infection with the HIV virus the host immune response is activated in an attempt to combat the infection and prevent the progression to AIDS. CD8+ T cells perform a crucial part of the host anti-HIV response in different ways. First, CD8+ cells from HIV infected individuals can recognize infected cells and respond by killing the cells lytically (CTL response). Typically, this response occurs during the initial phase of infection where a transient decrease in viremia can be seen. Also during the acute phase of infection, prior to antibody production, a further action of CD8+ T cells involves an innate, non-cytotoxic action, which results in a decrease of viral replication. This latter response is known as the CD8+ cell non-cytotoxic anti-HIV response (CNAR), and functions in a non-MHC restricted fashion (3, 4). While both of these activities are significant, the CNAR activity is of particular interest as it correlates with clinical status (5, 6). Specifically, long term HIV non-progressors, represented by infected but asymptomatic individuals who remain healthy without ever receiving anti-viral medication, display a heightened CNAR activity as compared to patients who display disease progression (7, 8).
CNAR is active against a wide variety of HIV strains and is able to suppress HIV replication in both CD4+ T cells as well as macrophages (9). Since its discovery in 1986, there has been keen interest by many research groups in understanding the molecular mechanism by which CNAR functions, however this remains incompletely understood. Several important studies have contributed to a partial characterization of CNAR.
It has been known for some time that the anti-HIV activity of CNAR results from the secretion of a proteinacious factor(s) from activated CD8+ T cells, and has been termed CAF for CD8+ anti-HIV factor (10). Interestingly, it has been shown that the secretion of an active HIV inhibitory CAF factor(s) is not limited to CD8+ T cells from HIV seropositive patients, but can also be detected in culture media from EBV specific T cell line (11). This result suggests that there may be a common or related CAF that could function to inhibit viral replication as a result of immune system activation.
Although several groups have identified proteins that display anti-HIV activity, none of these fulfill all the criteria that CAF is known to exhibit. Some examples of anti-HIV factors include alpha defensin, RANTES and other beta chemokines, the cell adhesion molecule VCAM, and bovine antithrombin III (12-15).
While the precise identity of CAF remains unknown, research by many groups has combined to reveal certain key features. Together with secretion from activated CD8+ T cells, studies have suggested that a proteolytic activity appears to be necessary for CAF activation since protease inhibitors can block CAF activity (16). To date, the responsible protease involved has not been identified. Mechanistically, it is known that CAF or a CAF-like activity can suppress viral replication by inhibiting HIV transcription driven by the viral LTR, but does not appear to function through inhibiting viral integration or reverse transcription (17-19). Further evidence suggests that the inhibition of viral transcription occurs via activation of the STAT1 signaling pathway (20). Given the observation that CAF activity functions through inhibition of viral LTR driven transcription, a mutational analysis of several transcription factor binding sites in the LTR has been performed (21). The results of this study indicate that CAF does not appear to rely on independent binding of NFAT, AP1, NF-κB or SP1 sites. With respect to viral transactivators, it is known that the HIV Tat transcription factor plays an essential role in viral replication through its interaction with the Tat transactivation response region (TAR). The TAR element is present both within the LTR as well as at the 5′ end of all viral transcripts where Tat interacts to activate transcription and promote efficient mRNA translation (22). The importance of the Tat/TAR system is highlighted by studies showing that impairing this interaction can dramatically inhibit HIV replication (23). The Tat/TAR system, perhaps together with other factors, is therefore a possible target for the effects of CAF.
Despite years of ongoing research in this area, there continues to be a need for new and improved medicines for treating and/or preventing HIV and AIDS. The present inventors have accordingly sought to identify new diagnostic and chemotherapeutic methods in this area by investigating the Tat/TAR system and potential CAF targets.
The present invention accordingly relates to compositions and methods for prevention and/or treatment of HIV infections, as well as compositions and methods to modulate HIV replication.
According to the present invention there is provided a polypeptide, protein or functional fragment thereof comprising an amino acid sequence that is between about 80% and about 100% identical to any one of the sequences of SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6; SEQ ID NO:7; SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10; SEQ ID NO:11; SEQ ID NO:12; SEQ ID NO:13; SEQ ID NO:14; SEQ ID NO:15; SEQ ID NO:16 or SEQ ID NO:17, or which comprises an amino acid sequence that is between about 80% and about 100% identical to the sequence of a deletion mutant of TOE1 including K335/D363; K335/D373, K335/D387; K335/D402; K335/D416; K335/D435; K335/D443; K335/S510; E329/D363; E329/D373; E329/D387; E329/D402; E329/D416; E329/D435; E329/D443; E329/S510; L323/D363; L323/D373; L323/D387; L323/D402; L323/D416; L323/D435; L323/D443; L323/S510; I321/D363; I321/D373; I321/D387; I321/D402; I321/D416; I321/D435; I321/D443; I321/S510; N302/D363; N302/D373; N302/D387; N302/D402; N302/D416; N302/D435; N302/D443; N302/S510; Y283/D435; Y283/D443; H280/D363; H280/D373; H280/D387; H280/D402; H280/D416; H280/D435; H280/D443; H280/S510; T229/D363; T229/D373; T229/D387; T229/D402; T229/D416; T229/D435; T229/D443; T229/S510; L218/D363; L218/D373; L218/D387; L218/D402; L218/D416; L218/D435; L218/D443; L218/S510; L196/D435; L196/D443; K111/D363; K111/D373; K111/D387; K111/D402; K111/D416; K111/D435; K111/D443; K111/S510; T65/D363; T65/D373; T65/D387; T65/D402; T65/D416; T65/D435; T65/D443; T65/S510; G18/D363; G18/D373; G18/D387; G18/D402; G18/D416; G18/D435; G18/D443; G18/S510; G8/D363; G8/D373; G8/D387; G8/D402; G8/D416; G8/D435; G8/D443; G8/S510; S5/D363; S5/D373; S5/D387; S5/D402; S5/D416; S5/D435; S5/D443; and S5/S510, wherein the indicated amino acids respectively specify the amino- and carboxy-terminal residues of the deletion mutants based on the sequence of SEQ ID NO:1, including fragments and variants thereof. In a further embodiment, the amino acid sequence is identical to the sequence of SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6; SEQ ID NO:7; SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10; SEQ ID NO:11; SEQ ID NO:12; SEQ ID NO:13; SEQ ID NO:14; SEQ ID NO:15; SEQ ID NO:16 or SEQ ID NO:17, or to the to the sequence of the following TOE1 mutants: K335/D363; K335/D373, K335/D387; K335/D402; K335/D416; K335/D435; K335/D443; K335/S510; E329/D363; E329/D373; E329/D387; E329/D402; E329/D416; E329/D435; E329/D443; E329/S510; L323/D363; L323/D373; L323/D387; L323/D402; L323/D416; L323/D435; L323/D443; L323/S510; I321/D363; I321/D373; I321/D387; I321/D402; I321/D416; I321/D435; I321/D443; I321/S510; N302/D363; N302/D373; N302/D387; N302/D402; N302/D416; N302/D435; N302/D443; N302/S510; Y283/D435; Y283/D443; H280/D363; H280/D373; H280/D387; H280/D402; H280/D416; H280/D435; H280/D443; H280/S510; T229/D363; T229/D373; T229/D387; T229/D402; T229/D416; T229/D435; T229/D443; T229/S510; L218/D363; L218/D373; L218/D387; L218/D402; L218/D416; L218/D435; L218/D443; L218/S510; L196/D435; L196/D443; K111/D363; K111/D373; K111/D387; K111/D402; K111/D416; K111/D435; K111/D443; K111/S510; T65/D363; T65/D373; T65/D387; T65/D402; T65/D416; T65/D435; T65/D443; T65/S510; G18/D363; G18/D373; G18/D387; G18/D402; G18/D416; G18/D435; G18/D443; G18/S510; G8/D363; G8/D373; G8/D387; G8/D402; G8/D416; G8/D435; G8/D443; G8/S510; S5/D363; S5/D373; S5/D387; S5/D402; S5/D416; S5/D435; S5/D443; and S5/S510, wherein the indicated amino acids are as described above.
Polypeptides as described herein will preferably involve purified or isolated polypeptide preparations. In certain embodiments, purification of the polypeptide may utilize recombinant expression methods well known in the art, and may involve the incorporation of an affinity tag into the expression construct to allow for affinity purification of the target polypeptide.
In addition, polypeptides as referred to herein may also be produced by synthetic preparative methods, for example but not limited to solid-phase synthetic processes.
Fragments of the above polypeptides are included herein, but are not limited to amino acid sequences wherein one or more amino acids are deleted. For example, but not to be considered limiting, a fragment may exist when one or more amino acids from the amino terminal, carboxy terminal or both are removed. Further, one or more amino acids internal to the polypeptide may be deleted.
Variants of the above polypeptides are also contemplated, and may comprise one or more amino acid substitutions, additions, insertions, or a combination thereof in the sequences shown herein. Preferably, the amino acid sequence exhibits greater than about 90% homology, more preferably greater than about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or about 100% homology to the sequence(s) described herein. The degree of homology may also be represented by a range defined by any two of the values listed above or any value therein between.
Amino acid substitutions, in a non-limiting embodiment, may include conservative amino acid substitutions. Conservative substitutions may mean substitutions with similarly charged amino acid residues or residues with similar polar properties. As an example, lysine (K) may be replaced with arginine (R), aspartic acid (D) may be replaced with glutamic acid (E). Other conservative amino acid replacements will be apparent to those skilled in the art.
Polypeptide variants as described herein may also refer to peptides having at least one modification. For instance, but not to be limiting, the modification may be carried out in a synthetic process such as cyclization, N terminus modification, C terminus modification, peptide bond modification including, but not limited to, CH2—NH, CH2—S, CH2—S—O, O═C—NH CH2-0, CH2—CH2, S═C—NH, CH═CH or CF═CH, backbone modification and residue modification. In other embodiments, polypeptide variants may encompass polypeptides prepared by recombinant means, and which are post-translationally modified. Methods for preparing such variants are well known in the art.
It is further contemplated that the amino acid sequence comprises greater than about 70%, more preferably about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or about 100% identity with the amino acid sequence(s) described herein. Further, the degree of identity may be represented by a range defined by any two of the values listed or any value therein between. Methods for determining % identity or % homology are known in the art and any suitable method may be employed for this purpose.
It is also envisioned that the above-described polypeptides, fragments and variants can be linked to a heterologous carrier molecule. In certain non-limiting embodiments, the heterologous carrier molecule may comprise a protein transduction domain. For example, yet without wishing to be limiting in any manner, the protein transduction domain may comprise the amino acid sequence of Tat (SEQ ID NO:18), or may comprise lipids or other chemical groups to induce cell permeability.
There is further provided an isolated polynucleotide which encodes the above-described polypeptide, protein or functional fragment thereof. Embodiments of such polynucleotides can be derived from the wild-type nucleotide sequence of TOE1, or may be obtained through reverse translation of the polypeptide sequences described herein.
The nucleotide sequences provided by the present invention may be part of a larger nucleotide sequence or nucleotide construct optionally comprising one or more regulatory sequences, for example promoters, terminators and the like. By the terms “regulatory sequence”, “regulatory region”, “regulatory element” it is meant a portion of nucleic acid typically, but not always, upstream of the protein or polypeptide coding region of a nucleotide sequence, which may be comprised of either DNA or RNA, or both DNA and RNA. When a regulatory region is active, and in operative association with a nucleotide sequence of interest, this may result in expression of the nucleotide sequence of interest. A regulatory element may be capable of mediating organ specificity, or controlling developmental or temporal nucleotide sequence activation. A “regulatory region” includes promoter elements, core promoter elements exhibiting a basal promoter activity, elements that are inducible in response to a stimulus, elements that mediate promoter activity such as negative regulatory elements or transcriptional enhancers. “Regulatory region”, as used herein, also includes elements that are active following transcription, for example, regulatory elements that modulate nucleotide sequence expression such as translational and transcriptional enhancers, translational and transcriptional repressors, upstream activating sequences, and mRNA instability determinants. Several of these latter elements may be located proximal to the coding region.
In further embodiments there are provided vectors comprising the above-described polynucleotides. The vector may be a plasmid, a cosmid, a phage, a virus, or a fragment of a virus. The vector can be an expression vector.
There is further provided a cell comprising a polynucleotide or vector as described above, as well as compositions comprising the polypeptide, polynucleotide or vector and a suitable carrier.
The above composition may, in an embodiment, be a pharmaceutical composition for treatment or prevention of HIV infection, or for modulating HIV replication, inhibiting HIV viral transcription, inhibiting the viral Tat protein, or inhibiting HIV LTR expression.
The above polypeptides and polynucleotides can be used in a method for treatment or prevention of human immunodeficiency virus (HIV) infection, for example by administering a polypeptide or composition as described herein to a subject in need of such treatment.
The above polypeptides and polynucleotides can also be used in a method to modulate the replication of HIV, inhibit viral transcription, inhibit the viral Tat protein, or inhibit HIV LTR expression. In certain embodiments the modulating of HIV replication comprises inhibiting of HIV replication. In addition, the modulating of HIV replication, inhibiting of HIV viral transcription, inhibiting of the viral Tat protein, or inhibiting of HIV LTR expression can be carried out either in vitro or in vivo.
In certain embodiments the human immunodeficiency virus is HIV-1 or HIV-2, or both.
A subject in the method(s) described herein may be a mammalian subject, for example, but not limited to mouse, cow, sheep, goat, pig, dog, cat, rat, rabbit, primate, or human. In an embodiment, which is not meant to be limiting, the subject is a human.
Also provided herein is a protein transduction domain comprising an amino acid sequence that is between about 80% and about 100% identical to the sequence of SEQ ID NO:15, or a fragment or variant thereof. In addition, there is provided an isolated nucleic acid encoding such a protein transduction domain, a vector comprising the nucleic acid encoding the protein transduction domain, a host cell transformed with the nucleic acid or vector encoding the protein transduction domain, or a composition comprising a therapeutically active molecule linked to the protein transduction domain. The therapeutically active molecule may be any molecule that has biological activity, such as but not limited to polypeptides, nucleic acids, organic compounds, and others as will be apparent to those skilled in the art.
In further embodiments, there is also provided an antibody which binds to the polypeptide described above, and in preferred embodiments, a polypeptide having the amino acid sequence of SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6; SEQ ID NO:7; SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10; SEQ ID NO:11; SEQ ID NO:12; SEQ ID NO:13; SEQ ID NO:14; SEQ ID NO:15; SEQ ID NO:16 or SEQ ID NO:17, or a fragment or variant thereof. The antibody may be, without intending to be limiting, either a monoclonal antibody or a polyclonal antibody. Such antibodies can be prepared using common techniques as will be apparent to those skilled in the art.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific products and procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following claims.
These and other features of the invention will become more apparent from the following description in which reference is made to the following figures:
The present invention provides a polypeptide comprising an amino acid sequence that is at least 80% identical to the sequence of TOE1 (SEQ ID NO: 1) or deletion mutants thereof including L196 (SEQ ID NO:2), L196/D416 (SEQ ID NO:3), L196/D402 (SEQ ID NO:4), L196/D387 (SEQ ID NO:5), L196/D373 (SEQ ID NO:6), L196/D363 (SEQ ID NO:7), Y283 (SEQ ID NO:8), Y283/D416 (SEQ ID NO:9), Y283/D402 (SEQ ID NO:10), Y283/D387 (SEQ ID NO:11), Y283/D373 (SEQ ID NO:12), Y283/D363 (SEQ ID NO:13), C300/R347 (SEQ ID NO:14), K335/R347 (SEQ ID NO:15), C300/D363 (SEQ ID NO:16) and K335/D363 (SEQ ID NO:17), or a fragment or variant of any one thereof. An alignment of the above-specified sequences can be seen in
The invention also provides a polypeptide comprising an amino acid sequence that is at least 80% identical to one of the following additional deletion mutants of TOE1: K335/D363; K335/D373, K335/D387; K335/D402; K335/D416; K335/D435; K335/D443; K335/S510; E329/D363; E329/D373; E329/D387; E329/D402; E329/D416; E329/D435; E329/D443; E329/S510; L323/D363; L323/D373; L323/D387; L323/D402; L323/D416; L323/D435; L323/D443; L323/S510; I321/D363; I321/D373; I321/D387; I321/D402; I321/D416; I321/D435; I321/D443; I321/S510; N302/D363; N302/D373; N302/D387; N302/D402; N302/D416; N302/D435; N302/D443; N302/S510; Y283/D435; Y283/D443; H280/D363; H280/D373; H280/D387; H280/D402; H280/D416; H280/D435; H280/D443; H280/S510; T229/D363; T229/D373; T229/D387; T229/D402; T229/D416; T229/D435; T229/D443; T229/S510; L218/D363; L218/D373; L218/D387; L218/D402; L218/D416; L218/D435; L218/D443; L218/S510; L196/D435; L196/D443; K111/D363; K111/D373; K111/D387; K111/D402; K111/D416; K111/D435; K111/D443; K111/S510; T65/D363; T65/D373; T65/D387; T65/D402; T65/D416; T65/D435; T65/D443; T65/S510; G18/D363; G18/D373; G18/D387; G18/D402; G18/D416; G18/D435; G18/D443; G18/S510; G8/D363; G8/D373; G8/D387; G8/D402; G8/D416; G8/D435; G8/D443; G8/S510; S5/D363; S5/D373; S5/D387; S5/D402; S5/D416; S5/D435; S5/D443; S5/S510, or a fragment or variant of any one thereof. The indicated amino acids respectively specify the amino- and carboxy-terminal residues of the deletion mutants as derived from the TOE1 sequence (SEQ ID NO:1).
In a preferred embodiment, the polypeptide is between about 15 and about 510 amino acids, for example, but not limited to 15, 17, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or 510 amino acids. However, the polypeptide length may also be defined by a range of any two of the values listed above. In addition, in an alternate embodiment, which is not meant to be limiting in any manner, the present invention contemplates polypeptides as defined above which comprises more than 510 amino acids.
A polypeptide may be of any length unless otherwise specified. A fragment is any polypeptide or nucleic acid that is shorter than its corresponding naturally occurring polypeptide or nucleic acid, respectively. A derivative is any polypeptide or nucleic acid that is altered with respect to a reference polypeptide or nucleic acid, respectively, and includes, for example fragments or mutants.
The present invention also contemplates polypeptides having an amino acid sequence that comprises 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino sequences described above. Further, the polypeptides may be defined as comprising a range of sequence identity defined by any two of the values listed above.
The present invention also provides a nucleic acid encoding polypeptides as defined above. For example, but not wishing to be limiting in any manner, the present invention contemplates a nucleic acid that encodes an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6; SEQ ID NO:7; SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10; SEQ ID NO:11; SEQ ID NO:12; SEQ ID NO:13; SEQ ID NO:14; SEQ ID NO:15; SEQ ID NO:16 or SEQ ID NO:17, or the K335/D363; K335/D373, K335/D387; K335/D402; K335/D416; K335/D435; K335/D443; K335/S510; E329/D363; E329/D373; E329/D387; E329/D402; E329/D416; E329/D435; E329/D443; E329/S510; L323/D363; L323/D373; L323/D387; L323/D402; L323/D416; L323/D435; L323/D443; L323/S510; I321/D363; I321/D373; I321/D387; I321/D402; I321/D416; I321/D435; I321/D443; I321/S510; N302/D363; N302/D373; N302/D387; N302/D402; N302/D416; N302/D435; N302/D443; N302/S510; Y283/D435; Y283/D443; H280/D363; H280/D373; H280/D387; H280/D402; H280/D416; H280/D435; H280/D443; H280/S510; T229/D363; T229/D373; T229/D387; T229/D402; T229/D416; T229/D435; T229/D443; T229/S510; L218/D363; L218/D373; L218/D387; L218/D402; L218/D416; L218/D435; L218/D443; L218/S510; L196/D435; L196/D443; K111/D363; K111/D373; K111/D387; K111/D402; K111/D416; K111/D435; K111/D443; K111/S510; T65/D363; T65/D373; T65/D387; T65/D402; T65/D416; T65/D435; T65/D443; T65/S510; G18/D363; G18/D373; G18/D387; G18/D402; G18/D416; G18/D435; G18/D443; G18/S510; G8/D363; G8/D373; G8/D387; G8/D402; G8/D416; G8/D435; G8/D443; G8/S510; S5/D363; S5/D373; S5/D387; S5/D402; S5/D416; S5/D435; S5/D443; or S5/S510 deletion mutants of TOE1 described above, or a fragment or variant of any one thereof.
By “percent identical” or “percent identity”, it is meant one or more than one nucleic acid or amino acid sequence that is substantially identical to a coding sequence or amino acid sequence of the specified polypeptide. By “substantially identical” is meant any nucleotide or amino acid sequence with similarity to the sequence of a nucleic acid or amino acid of the invention, or a fragment or a derivative thereof. The term “substantially identical” can also be used to describe similarity of polypeptide sequences. For example, nucleotide sequences or to polypeptide sequences that are at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 98% or 99% identical to the polypeptide coding sequence, or the encoded polypeptide, respectively, or fragments or derivatives thereof, and still retain ability to affect HIV replication are contemplated.
To determine whether a nucleic acid exhibits identity with the sequences presented herein, oligonucleotide alignment algorithms may be used, for example, but not limited to a BLAST (GenBank URL: www.ncbi.nlm.nih.gov/cgi-bin/BLAST/, using default parameters: Program: blastn; Database: nr; Expect 10; filter: default; Alignment: pairwise; Query genetic Codes: Standard(1)), BLAST2 (EMBL URL: http://www.embl-heidelberg.de/Services/index.html using default parameters: Matrix BLOSUM62; Filter: default, echofilter: on, Expect: 10, cutoff: default; Strand: both; Descriptions: 50, Alignments: 50), or FASTA search, using default parameters. Polypeptide alignment algorithms are also available, for example, without limitation, BLAST 2 Sequences (www.ncbi.nlm.nih.gov/blast/bl2seq/bl2.html, using default parameters Program: blastp; Matrix: BLOSUM62; Open gap (11) and extension gap (1) penalties; gap x_dropoff: 50; Expect 10; Word size: 3; filter: default).
An alternative indication that two nucleic acid sequences are substantially identical is that the two sequences hybridize to each other under moderately stringent, or preferably stringent, conditions. Hybridization to filter-bound sequences under moderately stringent conditions may, for example, be performed in 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.2×SSC/0.1% SDS at 42° C. for at least 1 hour (see Ausubel, et al. (eds), 1989, Current Protocols in Molecular Biology, Vol. 1, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3). Alternatively, hybridization to filter-bound sequences under stringent conditions may, for example, be performed in 0.5 M NaHPO4, 7% SDS, 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C. for at least 1 hour (see Ausubel, et al. (eds), 1989, supra). Hybridization conditions may be modified in accordance with known methods depending on the sequence of interest (see Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2 “Overview of principles of hybridization and the strategy of nucleic acid probe assays”, Elsevier, N.Y.). Generally, but not wishing to be limiting, stringent conditions are selected to be about 5° C. lower than the thermal melting point for the specific sequence at a defined ionic strength and pH.
By protein transduction domain it is meant a sequence of nucleic acids that encode a polypeptide, or a sequence of amino acids comprising the polypeptide, wherein the polypeptide facilitates localization to a particular site, for example a cell or the like, or it may facilitate transport across a membrane or lipid bilayer. The polypeptides and nucleic acids of the present invention may be fused to a protein transduction domain to facilitate transit across lipid bilayers or membranes.
Many polypeptides and nucleic acids do not efficiently cross the lipid bilayer of the plasma membrane, and therefore enter into cells at a low rate. However, there are certain naturally occurring polypeptides that can transit across membranes independent of any specific transporter. Antennapedia (Drosophila), Tat (HIV) and VP22 (Herpes) are examples of such polypeptides. Fragments of these and other polypeptides have been shown to retain the capacity to transit across lipid membranes in a receptor-independent fashion. These fragments, termed protein transduction domains, are generally 10 to 27 amino acids in length, possess multiple positive charges, and in several cases have been predicted to be amphipathic. Polypeptides and nucleic acids that are normally inefficient or incapable of crossing a lipid bilayer can be made to transit the bilayer by being fused to a protein transduction domain.
U.S. Publication 2002/0142299 (which is incorporated herein by reference) describes a fusion of Tat with human beta-glucuronidase. This fusion protein readily transits into various cell types both in vitro and in vivo. Furthermore, Tat fusion proteins have been observed to cross the blood-brain-barrier. Frankel et al. (U.S. Pat. No. 5,804,604, U.S. Pat. No. 5,747,641, U.S. Pat. No. 5,674,980, U.S. Pat. No. 5,670,617 and U.S. Pat. No. 5,652,122; which are incorporated herein by reference) have also demonstrated transport of a protein (beta-galactosidase or horseradish peroxidase) into a cell by fusing the protein with amino acids 49-57 of Tat.
PCT Publication WO01/15511 (which is incorporated herein by reference) discloses a method for developing protein transduction domains using a phage display library. The method comprises incubating a target cell with a peptide display library and isolating internalized peptides from the cytoplasm and nuclei of the cells and identifying the peptides. The method further comprises linking the identified peptides to a protein and incubating the peptide-protein complex with a target cell to determine whether uptake is facilitated. Using this method a protein transduction domain for any cell or tissue type may be developed. US Publication 2004/0209797 (which is incorporated herein by reference) shows that reverse isomers of several of the peptides identified by the above can also function as protein transduction domains.
PCT Publication WO99/07728 (which is incorporated herein by reference) describes linearization of protegrin and tachyplesin, naturally occurring as a hairpin type structure held by disulphide bridges. Irreversible reduction of disulphide bridges generated peptides that could readily transit cell membranes, alone or fused to other biological molecules. US Publication 2003/0186890 (which is incorporated herein by reference) describes derivatives of protegrin and tachyplesin that were termed SynB1, SynB2, SynB3, etc. These SynB peptides were further optimized for mean hydrophobicity per residue, helical hydrophobic moment (amphipathicity), or beta hydrophobic moment. Various optimized amphipathic SynB analog peptides were shown to facilitate transfer of doxorubicin across cell membranes. Further, doxorubicin linked to a SynB analog was observed to penetrate the blood-brain-barrier at 20 times the rate of doxorubicin alone.
The protein transduction domains described in the preceding paragraphs are only a few examples of the protein transduction domains available for facilitating membrane transit of small molecules, polypeptides or nucleic acids. Other examples are transportan, W/R, AlkCWK18, DipaLytic, MGP, or RWR. Further examples include lipids and other chemical groups to induce cell permeability. Still many other examples will be recognized by persons skilled in the art.
A protein transduction domain and a polypeptide of the present invention may be placed together in sufficient proximity and maintained together for a sufficient time to allow the protein transduction domain to influence pharmaceutical product performance of the polypeptide. Contemplated associations of protein transduction domain and polypeptide include, for example and without limitation: non-covalent associations such as electrostatic interactions, hydrogen bonding, ionic bonds or complexes, Van der Waals bonds; covalent linkages such as conventional methods of cross-linking; linkages that are activated, in vitro and/or in vivo by electromagnetic radiation; any covalent bond such as a peptide bond; any biochemical interaction known to protein biochemists, such as biotin/streptavidin, nickel/Histidine, glutathione/glutathione-S-transferase, or antigen/antibody; physical associations within matrix structures or encapsulating systems; etc.
A polypeptide of the invention can be synthesized in vitro or delivered to a cell in vivo by any conventional method. As a representative example of an in vitro method, the polypeptide may be chemically synthesized in vitro, or may be enzymatically synthesized in vitro in a suitable biological expression system, such as without limitation, wheat germ extract or rabbit reticulocyte lysate. As a representative example of an in vivo method, a DNA, RNA, or DNA/RNA hybrid molecule comprising a nucleotide sequence encoding a polypeptide of the invention is introduced into an animal, and the nucleotide sequence is expressed within a cell of an animal.
The nucleotide sequence may be operably linked to regulatory elements in order to achieve preferential expression at desired times or in desired cell or tissue types. Furthermore, as will be known to one of skill in the art, other nucleotide sequences including, without limitation, 5′ untranslated regions, 3′ untranslated regions, cap structures, poly A tail, translational initiators, sequences encoding signaling or targeting peptides, translational enhancers, transcriptional enhancers, translational terminators, transcriptional terminators, transcriptional promoters, may be operably linked with the nucleotide sequence encoding a polypeptide (see as a representative examples “Genes VII”, Lewin, B. Oxford University Press (2000) or “Molecular Cloning: A Laboratory Manual”, Sambrook et al., Cold Spring Harbor to Laboratory, 3rd edition (2001)). A nucleotide sequence encoding a polypeptide or a fusion polypeptide comprising a polypeptide agent and a protein transduction domain may be incorporated into a suitable vector. Vectors may be commercially obtained from companies such as Stratagene or InVitrogen. Vectors can also be individually constructed or modified using standard molecular biology techniques, as outlined, for example, in Sambrook et al. (Cold Spring Harbor Laboratory, 3rd edition (2001)). A vector may contain any number of nucleotide sequences encoding desired elements that may be operably linked to a nucleotide sequence encoding a polypeptide or fusion polypeptide comprising a protein transduction domain. Such nucleotide sequences encoding desired elements, include, but are not limited to, transcriptional promoters, transcriptional enhancers, transcriptional terminators, translational initiators, translational, terminators, ribosome binding sites, 5′ untranslated regions, 3′ untranslated regions, cap structures, poly A tail, origin of replication, detectable markers, affinity tags, signals or target peptides. Persons skilled in the art will recognize that the selection and/or construction of a suitable factor may depend upon several factors, including, without limitation, the size of the nucleic acid to be incorporated into the vector, the type of transcriptional and translational control elements desired, the level of expression desired, copy number desired, whether chromosomal integration is desired, the type of selection process that is desired, or the host cell or the host range that is intended to be transformed.
The DNA, RNA, or DNA/RNA hybrid molecule may be introduced intracellularly, extracellularly into a cavity, interstitial space, into the circulation of an organism, orally, or by any other standard route of introduction for therapeutic molecules and/or pharmaceutical compositions. Standard physical methods of introducing nucleic acids include, but are not limited to, injection of a solution comprising RNA, DNA, or RNA/DNA hybrids, bombardment by particles covered by the nucleic acid, bathing a cell or organism in a solution of the nucleic acid, or electroporation of cell membranes in the presence of the nucleic acid.
A nucleic acid may be introduced into suitable eukaryotic cells ex vivo and the cells harboring the nucleic acid can then be inserted into a desired location in an animal. A nucleic acid can also be used to transform prokaryotic cells, and the transformed prokaryotic cells can be introduced into an animal, for example, through an oral route. Those skilled in the art will recognize that a nucleic acid may be constructed in such a fashion that the transformed prokaryotic cells can express and secrete a polypeptide of the invention. Preferably, the prokaryotic cell is part of the animal's endogenous intestinal microflora. With regards to human examples of endogenous microflora are, without wishing to be limiting, Lactobacillus acidophillus, Streptococcus thermophilus, and Bifidobacterium bifidum. A nucleic acid may also be inserted into a viral vector and packaged into viral particles for efficient delivery and expression.
A polypeptide of the present invention, or nucleic acids encoding these polypeptides, may be formulated into any convenient dosage form. The dosage form may comprise, but is not limited to an oral dosage form wherein the agent is dissolved, suspended or the like in a suitable excipient such as but not limited to water. In addition, the agent may be formulated into a dosage form that could be applied topically or could be administered by inhaler, or by injection either subcutaneously, into organs, or into circulation. An injectable dosage form may include other carriers that may function to enhance the activity of the agent. Any suitable carrier known in the art may be used. Also, the polypeptide may be formulated for use in the production of a medicament. Many methods for the productions of dosage forms, medicaments, or pharmaceutical compositions are well known in the art and can be readily applied to the present invention by persons skilled in the art.
It is also envisioned that carrier molecules or groups may be incorporated into the polypeptide of the present invention, or nucleic acids encoding these polypeptides, or a formulation, composition or medicament ad described herein, to enhance permeability through the brain-blood-barrier. Such carrier molecules or groups will be apparent and can be varied according to the knowledge of those skilled in the relevant art.
Combination therapy with polypeptides of the present invention or other agents useful for preventing and/or treating HIV infection or replication is also contemplated. With regards to combination therapy, suitable dosage forms again include capsules, tablets, and the like, preferably for oral administration, although any dosage form, for any route of administration is contemplated. Combination therapy can be administered as separate entities, e.g. two tablets or other forms, each containing one agent, or may be administered as a single dosage form containing both drugs, or concomitant use.
In case of oral administration of two or more different agents, the single dose can be, but is not limited to a capsule, tablet, or oral solution, and it may also contain inactive component(s) necessary to form the single delivery system.
Combination therapy medications of the present invention may be administered by any desired route without limitation, for example administration can be transdermal (patch), buccal, sublingual, topical, nasal, parenteral (subcutaneous, intramuscular, intravenous, intradermal), rectal, or vaginal. Various combinations of controlled release/rapid release are also contemplated.
The methods and compositions of the present invention are useful for preventing and/or treating HIV infection or replication. These will be further illustrated in the following experiments.
The TOE1 gene has been previously cloned, as a genetic target of the Egr1 transcription factor using a chromatin immunoprecipitation technology (24), and was also shown to be upregulated in CD8+ T cells (25). Specifically, TOE1 was among three identified transcripts that were found upregulated in the CD8+ population derived from an HIV-infected long term non-progressor, in comparison with his uninfected twin sibling. To better understand the biological role of TOE1 the present inventors have studied its importance in CNAR and found that TOE1, and derivatives thereof as described herein, inhibits HIV replication.
If TOE1 functions in a CAF-like manner, then it should affect viral LTR driven transcription. Interestingly, a structural comparison between TOE1 and Tat revealed a region of homology between these two proteins (
To test for a potential interaction between TOE1 and Tat, we assessed the effect of TOE1 expression on Tat driven transactivation of the TAR element. We employed a luciferase expression system controlled by the HIV LTR including the TAR sequence. As shown in
During characterization of TOE1 we observed multiple bands by Western blot analysis suggesting potential proteolytic processing. Since, as described earlier, CAF activity is reported to be dependent upon proteolytic activity, we decided to explore the possibility that TOE1 is a protease substrate. Using the ExPASy (Expert Protein Analysis System) protemics peptide cutter prediction software, we found that TOE1 is predicted to be a substrate for the pro-inflammatory protease ICE (caspase-1). The software predicts a single cleavage for the tetrapeptide FTAD at position 214-217 of TOE1. We considered this potential cleavage to warrant further study for several reasons. First, CAF is associated with viral infection, which is known to activate the inflammatory response, including caspase-1 activation. Secondly, very few biological substrates for caspase-1 are currently known, so the identification of a new substrate for this enzyme is of great interest for inflammation and cell death. To test this possibility, we isolated TOE1 by immunoprecipitation from TOE1 expressing THP1 cells using an affinity purified antibody against the C-terminal of TOE1, and then performed in vitro digestion with recombinant purified caspase-1 (Calbiochem). We found that TOE1 was indeed processed by caspase-1, and generated multiple cleaved forms. Significantly, processing was inhibited by a specific inhibitor of caspase-1, YVAD-CHO (
As caspase cleavage sites are invariant aspartates at the C termini of tetrapeptide recognition motifs, we generated a series of TOE1 constructs bearing mutations in asparate residues changed to alanines. In constructing mutants we considered candidate caspase-1 cleavage sites predicted on the basis of the fragments size observed by in vitro caspase-1 cleavage (see
We then tested whether preventing processing by caspase-1 could alter TOE1s effect on Tat transactivation of HIV LTR. As shown in
TOE1 is Secreted from THP1 Cells Following an Inflammatory Stimulus and α-CD3-Stimulated Human CD8+ Cells
Without wishing to be limited by theory, it is thought that TOE1 might exhibit CAF-like activity. Since CAF acts through a secretion/uptake pathway, we tested whether we could witness any secretion of TOE1. To this end, we treated THP1 cells with LPS to provide a proinflammatory stimulus and to activate caspase-1 in these cells. We found that TOE1, despite its nucleolar localization, was actively secreted in the culture medium beginning at 1 hour following the inflammatory stimulus. Both full-length and smaller TOE1 species, consistent with the products of previously observed caspase-1 cleavage, were found secreted into the culture medium (
In view of the finding that TOE1 is a substrate for caspase-1, it is believed that processing by caspase-1 may be implicated in its inhibitory role in HIV LTR transcription. As a result, it will be useful to define the minimal active TOE1 fragment generated by caspase-1. By identifying this region of TOE1 a further understanding the structural features of TOE1 participating in Tat inhibition can be achieved. This information can also be used for the design of therapeutic compounds.
As described earlier, we found that a double mutant of TOE1 that is not cleaved by caspase-1 at aspartates 195 and 282 has a reduced inhibitory activity of Tat-dependent transcription. Furthermore, we found that a deletion mutant of TOE1, delN282, displays a more powerful inhibitory activity. As can be seen in
Each of the aspartate residues cleaved by caspase-1 can be identified as shown above for the mapping of the D195 and D282 cleavage sites. Specifically, mutagenesis of TOE1 was performed by changing the aspartates at all candidate sites into alanines using QuickChange mutagenesis, developed by Stratagene. To this end, we have successfully identified additional caspase-1 cleavage sites corresponding to TOE1 amino acids 363 and 373 that are responsible for generating an approximately 17 kDa proteolytic fragment (
To determine if TOE1 could be secreted and subsequently taken up by other neighboring cells, we performed co-culture experiments of cells transfected with the HIV LTR reporter and Tat protein in one cell type and either full length TOE1 or the potential protein transduction domain of TOE1 comprising amino acids 283-363 in another cell type. As can be seen in
Active TOE1 fragments can be tested in vivo for their ability to block HIV replication in primary human cells. This can be carried out by making a GST fusion construct of the minimal active TOE1 fragment to be amplified and expressed in bacteria. This GST fusion protein can then be extracted and purified through Glutathione Sepharose chromatography. The GST tag can be cleaved by incubation of GST protein, bound to Glutathione Sepharose, with a biotinylated thrombin that is subsequentially removed by capture with Streptavidin Sepharose and centrifugation. The purified recombinant TOE1 active fragment can then be dialyzed against PBS, and sterile filtered for in vivo testing as described below.
Peripheral blood is obtained from normal healthy donors and peripheral blood mononuclear cells (PBMCs) prepared by centrifugation on a Ficoll-Hypaque density gradient. Next, CD4+ T cells are isolated by using a negative selection kit according to the manufacturer's instructions (StemCell Technologies), activated with PHA-L (1 μg/ml) and maintained in complete culture medium supplemented with IL-2 (30 U/ml) at a density of 2×106 cells/ml. Experiments can be conducted with different fully infectious laboratory (i.e. NL4-3, NL4-3-Balenv, JR-CSF) and primary isolates of HIV-1 bearing distinct co-receptor utilization profiles (i.e. R5/macrophage-tropic, X4/T-tropic, and R5X4/dual-tropic). Such HIV-1 variants can be obtained through the NIH AIDS Research and Reference Reagent Program (Germantown, Md.). The luciferase-encoding pNL4-3Luc+ E− R+ vector can be used to generate single-cycle luciferase reporter viruses pseudotyped with the broad host range vesicular stomatitis virus envelope glycoprotein G (VSV-G). Virus stocks can be prepared by transient transfection of 293T cells. Primary isolates of HIV-1 are expanded in human PBMCs. Each virus preparation is filtered through a 0.22 μm cellulose acetate syringe filter and purified through ultracentrifugation. This step eliminates free p24, the presence of which can lead to an overestimation of the actual number of infectious viral entities. Virus stocks are normalized for virion content using an in-house sensitive double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) specific for the viral p24 protein. Activated CD4+ T cells are pulsed with HIV-1 viruses (10 ng of p24/105 cells) for two hours, washed, and then incubated with various concentrations of recombinant TOE1 active fragment prepared as described above. Virus production will be assessed at 2, 4 and 6 days post-infection by measuring the p24 levels in cell-free culture supernatants. All measurements will be performed in triplicate. Infection with luciferase-encoding viruses will be assessed at 3 days post-infection by measuring luciferase activity. The use of such single-cycle luciferase reporter viruses will allow us to measure the effect of TOE1 specifically on HIV-1 expression.
Experiments can be conducted with fully infectious viruses where the recombinant TOE1 is added before infection, and at various time points post-infection. This allows for the analysis of the effects of TOE1 on different steps on the viral life cycle i.e. viral entry/fusion, reverse transcription and integration, and viral expression.
The TOE1 inhibitory activity on the HIV LTR can also be studied in primary CD4+ T cells. To this end, resting or activated CD4+ T cells are transfected with HIV LTR driven luciferase encoding construct alone or along with the Tat expression vector pCEP4−Tat by Nucleofection using the human T cell nucleofector kit from Amaxa (now Lonza). Transfected cells are then incubated with recombinant TOE1 active fragment, and luciferase activity is measured, e.g. at 6, 24 and 48 hours post transfection. The effect of the TOE1 polypeptide can also be tested in other primary human cells including monocyte-derived macrophages (MDMs) and immature monocyte-derived dendritic cells (iMDDCs). In brief, CD14+ cells are isolated by using a CD14 positive selection kit (MACS CD14 micro beads from StemCell Technologies). To generate iMDDC, purified monocytes are cultured in RPMI 1640/10% fetal calf serum supplemented with granulocyte-macrophage colony stimulating factor (GM-CSF) (1,000 U/ml) and interleukin-4 (IL-4) (200 U/ml) for 7 days. To generate MDMs, monocytes purified by adherence to plastic are allowed to differentiate into macrophages in culture medium supplemented with recombinant human macrophage colony stimulating factor, at a final concentration of 100 ng/mL for 6 days. Infection assays are performed essentially as described above for CD4+ T cells, using R5-tropic viruses such as NL4-3BalEnv and VSVG-pseudotyped viruses.
One or more currently preferred embodiments have been described by way of example. It will be apparent to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as defined in the claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2695337 | Mar 2010 | CA | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/CA2011/000234 | 3/4/2011 | WO | 00 | 11/26/2012 |
