US20090181458A1

US20090181458A1 - Tubulo-vesicular structure localization signals

Info

Publication number: US20090181458A1
Application number: US11/999,143
Authority: US
Inventors: Thomas David Reed
Original assignee: Intrexon Corp
Current assignee: Precigen Inc
Priority date: 2006-12-04
Filing date: 2007-12-04
Publication date: 2009-07-16
Also published as: US20110195504A1; US8993742B2

Abstract

The invention relates to polarized cell tubulo-vesicular structure localization signals. The localization signals are utilized as research tools or are linked to polypeptides of interest or therapeutic molecules. Disclosed are methods of making and using polypeptides and modified polypeptides as signals to localize therapeutics, experimental compounds, peptides, proteins and/or other macromolecules to the tubulo-vesicular structures of polarized cells. The polypeptides of the invention optionally include linkage to reporters, epitopes and/or other experimental or therapeutic molecules. The invention also encompasses polynucleotides encoding the localization signals and vectors comprising these polynucleotides.

Description

This application claims benefit of priority to U.S. 60/868,538, filed 4 Dec. 2006.

FIELD OF INVENTION

The invention relates to subcellular localization signals. In particular, the invention relates to tubulo-vesicular structure localization signals in monomeric form. The localization signals are utilized as research tools or are linked to therapeutics.
This application has subject matter related to application Ser. Nos. 10/682,764 (US2004/0185556, PCT/US2004/013517, WO2005/040336), 11/233,246, and US20040572011P (WO2005116231). Each of these patents and applications is hereby incorporated by reference.

BACKGROUND AND PRIOR ART

Drugs that act intracellularly generally enter cells by diffusion. Most drugs are small molecules because they have the ability to diffuse across tubulo-vesicular structures or organelle membranes to reach their site of action. To increase the bioavailability of a drug, often small molecules must be modified and/or formulated for greater solubility and/or permeability, depending on route of administration. Even small diffusible drugs may not be efficacious at their site of action. For example, multidrug resistance (MDR) may be present, which results in active efflux of drugs that enter cells with MDR. MDR often occurs in cancer cells.
In contrast to small molecules, high molecular weight compounds and polymer drugs, such as polynucleotides, polypeptides, and other macromolecules have little to no ability to diffuse across membranes. High molecular weight material is generally internalized by endocytosis. The addition of affinity binding partners to high molecular weight material can direct the high molecular weight compound to specific cells, and thereby result in increased selective uptake. However, once endocytosed, the material still remains separated from the cellular cytoplasm by a biological membrane.
Furthermore, endocytosed material is often delivered to the lysosome, where material sensitive to lysosomal enzymes is quickly degraded if steps are not taken to protect its breakdown or to facilitate escape from the lysosome. Delivery of high molecular weight compounds to their site of action at effective levels is a problem. It is therefore desirable to improve delivery to a desired subcellular compartment.
An aspect of the invention is to provide novel tubular vesicular structure localization signals by modifying one or more proteins capable of locating to the tubulo-vesicular structure of polarized cells by truncation or by amino acid substitution. Truncations, amino acid substitutions, and other modifications of known tubulo-vesicular structure-locating proteins are made to minimize endogenous biological activities other than localization. In general, the invention relates to cellular localization signals. More specifically, the invention relates to tubulo-vesicular structure localization signals in polarized cells. In polarized cells, such as kidney epithelial cells, which contain membranes, localization signals are selective for one of these tubulo-vesicular structure locations.
The polarized cell tubulo-vesicular structure localization signals are monomeric units that can be used separately or together for the purpose of targeting a polypeptide or other molecule of interest to a desired tubulo-vesicular structure location. Monomeric units used together exploit cooperation and synergism among individual signals in order to enhance strength and/or performance of individual signals. For example, some localization signals function at the N-terminus of a polypeptide of interest, and other localization signals function at the C-terminus of a polypeptide of interest. The signals of this invention encompass localization signals placed toward the N-terminus or C-terminus, or both the N-terminus and C-terminus of a polypeptide or other molecule of interest.
The localization signals are utilized as research tools or are linked to therapeutics. Disclosed are methods of making and using polypeptides and modified polypeptides as signals to localize therapeutics, experimental compounds, peptides, proteins and/or other macromolecules to the tubulo-vesicular structure of polarized eukaryotic cells, such as kidney epithelial cells. The polypeptides of the invention optionally include linkage to reporters, epitopes and/or other experimental or therapeutic molecules. The invention also encompasses polynucleotides encoding the localization signals and vectors comprising these polynucleotides.

DETAILED DESCRIPTION OF POLYPEPTIDE SEQUENCES

SEQ ID NOS:1-4 are full length sequences of proteins that localize to the tubulo-vesicular structure of polarized cells. These sequences have the following public database accession numbers: AAB23443, AAA40607, CAA62566, AAA40602. Each of the sequences represented by these accession numbers is incorporated by reference herein.
Specifically, the polypeptide of SEQ ID NO:1 is a neuronal dopamine transporter, which locates to the tubulo-vesicular structure in Madin-Darby Canine Kidney (MDCK) polarized cells when expressed therein. The polypeptide of SEQ ID NO:2 is a neuronal GABA transporter 3, which locates to the tubulo-vesicular structure in MDCK polarized cells when expressed therein. The polypeptide of SEQ ID NO:3 is a neuronal norepinephrine transporter, which locates to the tubulo-vesicular structure in MDCK polarized cells when expressed therein. The polypeptide of SEQ ID NO:4 is a neuronal GABA transporter 2, which locates to the tubulo-vesicular structure in MDCK polarized cells when expressed therein.
SEQ ID NOS:5-8 represent examples of monomeric tubulo-vesicular structure localization signals. SEQ ID NOS:5-8 are subsequences of SEQ ID NOS:1-4, which represent examples of peptide sequences that confer tubulo-vesicular structure localization. SEQ ID NO:5 and SEQ ID NO:7 represent examples of polypeptides useful as apical and basolateral localization signals, respectively, when linked to the N-terminus of a polypeptide or other molecule of interest. SEQ ID NO:6 and SEQ ID NO:8 represent examples of polypeptides useful as apical and basolateral localization signals, respectively, when linked to the C-terminus of a polypeptide or other molecule of interest.

DETAILED DESCRIPTION OF DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIGS. 1A-1F show examples of localization signals linked to an experimental or therapeutic polypeptide of interest.

FIGS. 2A-2G show examples of localization signals linked to an epitope tag, and an experimental or therapeutic polypeptide of interest.

FIGS. 3A-3F show examples of localization signals linked to a reporter.

FIGS. 4A-4H show examples of gene constructs where localization signals are linked to an experimental or therapeutic polypeptide of interest, with an optional epitope tag and/or reporter.

FIGS. 5A-5D show examples of vectors containing tubulo-vesicular structure localization signal gene constructs.

FIGS. 6-7 show examples of vectors for expression of tubulo-vesicular structure localization signals.

FIGS. 8-11 and 17-19 show additional examples of vectors for expression of tubulo-vesicular structure localization signals optionally under the control of the RheoSwitch inducible gene expression system.

FIGS. 12-16 show examples of microscopic images, which demonstrate the expression of tubulo-vesicular structure localization signals localized to the tubulo-vesicular structure.

BRIEF DESCRIPTION OF THE INVENTION

The invention relates to polarized cell tubulo-vesicular structure localization signals. Various embodiments of the tubulo-vesicular structure localization signals are represented in SEQ ID NOS:5-8. More specifically, the invention relates to monomeric localization signals used toward either the N-terminus of a polypeptide, the C-terminus of a polypeptide, or both the N-terminus and C-terminus of a polypeptide. Additionally, the invention relates to monomeric tubulo-vesicular structure localization signals comprising one or more subsequences of SEQ ID NOS:1-4 or any portion thereof. Furthermore, the invention relates to monomeric localization signals with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% and 99% sequence identity to a polypeptide comprising one or more of SEQ ID NOS:5-8 or any portion thereof. Furthermore, the invention relates to monomeric localization signals with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% and 99% sequence identity to a polypeptide comprising one or more subsequences of SEQ ID NOS:1-4.
A monomeric tubulo-vesicular structure localization signal is a polypeptide where at least a portion of the polypeptide is capable of conferring tubulo-vesicular structure location in polarized cells. A tubulo-vesicular structure localization signal functions to direct a polypeptide or other molecule of interest to the tubulo-vesicular structure.
A monomeric apical tubulo-vesicular structure localization signal functions to direct a polypeptide or other molecule of interest to the tubulo-vesicular structure of a polarized cell.
A monomeric tubulo-vesicular structure localization signal functions to direct a polypeptide or other molecule of interest to the tubulo-vesicular structure of a polarized cell.
One embodiment of the invention is a monomeric polarized cell tubulo-vesicular structure localization signal containing a polypeptide at least 80% identical to one of SEQ ID NOS:5-8.
Another embodiment of the invention is a monomeric localization signal containing a polypeptide at least 80% identical to one of SEQ ID NO:5 or SEQ ID NO:6.
Another embodiment of the invention is a combination localization signal containing polypeptides at least 80% identical to SEQ ID NO:5 and SEQ ID NO:6, wherein SEQ ID NO:5 is placed toward the N-terminus of a polypeptide of interest, and wherein SEQ ID NO:6 is placed toward the C-terminus of a polypeptide of interest.
Another embodiment of the invention is a monomeric localization signal containing a polypeptide at least 80% identical to one of SEQ ID NO:7 or SEQ ID NO:8.
Another embodiment of the invention is a combination localization signal containing polypeptides at least 80% identical to SEQ ID NO:7 and SEQ ID NO:8, wherein SEQ ID NO:7 is placed toward the N-terminus of a polypeptide of interest, and wherein SEQ ID NO:8 is placed toward the C-terminus of a polypeptide of interest.
Another embodiment of the invention is a localization signal containing a polypeptide at least 80% identical to one or more subsequences of SEQ ID NOS:1-4.
The localization signals of the invention are optionally linked to additional molecules or amino acids that provide an epitope, a reporter, and/or an experimental or therapeutic molecule. The epitope and/or reporter and/or experimental molecule and/or therapeutic molecule may be the same molecule. The epitope and/or reporter and/or experimental molecule and/or therapeutic molecule may also be different molecules. Experimental or therapeutic molecules include but are not limited to proteins and polypeptides.
The invention also encompasses polynucleotides comprising nucleotide sequences encoding tubulo-vesicular structure localization signals. The nucleic acids of the invention are optionally linked to additional nucleotide sequences encoding polypeptides with additional features, such as an epitope, a reporter, an experimental and/or therapeutic molecule. The polynucleotides are optionally flanked by nucleotide sequences comprising restriction endonuclease sites and other nucleotides needed for restriction endonuclease activity. The flanking sequences optionally provide unique cloning sites within a vector and optionally provide directionality of subsequence cloning. Further, the nucleic acids of the invention are optionally incorporated into vector polynucleotides. The localization signals of this invention have utility in compositions for research tools and/or therapeutics.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to localization signals. Various embodiments of the localization signals are represented by SEQ ID NOS:5-8 or additional partial sequences of SEQ ID NOS:1-4. An example of a monomeric localization signal is the polypeptide represented by SEQ ID NO:5. SEQ ID NO:5 is a selected subsequence of wild type full length SEQ ID NO:1. Another example of a monomeric localization signal is the polypeptide represented by SEQ ID NO:6. Each of SEQ ID NOS:5-8 represents an individual tubulo-vesicular structure localization signal in monomeric form. SEQ ID NOS:5-8 are selected examples of subsequences of SEQ ID NOS:1-4, however, other subsequences of SEQ ID NOS:1-4 may also be utilized as monomeric localization signals. Monomeric subsequences of SEQ ID NOS:1-4 may be wild type subsequences. Additionally, monomeric subsequences of SEQ ID NOS:1-4 may have some amino acids different than the wild type parent. Furthermore, monomeric localization signals may have 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a polypeptide comprising one or more of SEQ ID NOS:5-8. Furthermore, monomeric localization signals may have 80%, 85%, 90%, 95%, 96%, 97%, 98% and 99% sequence identity to a subsequence of SEQ ID NOS:1-4.
Monomeric localization signals may be utilized separately or in combination with each other within a polypeptide of interest to be targeted to the membrane (FIGS. 1A-1F). Monomeric units used in combination exploit synergism among individual signals in order to enhance performance of the intended localization. For example, some localization signals provide function when placed toward the N-terminus of a polypeptide of interest, and other localization signals provide function when placed toward the C-terminus of a polypeptide of interest. Therefore, SEQ ID NO:5 placed at the N-terminus of a polypeptide of interest may be used in conjunction with SEQ ID NO:6 placed at the C-terminus of a polypeptide of interest to enhance performance of either signal used separately to target the polypeptide to the tubulo-vesicular structure of polarized cells. Likewise, SEQ ID NO:7 and SEQ ID NO:8 when placed at the N-terminus and C-terminus, respectively, of a polypeptide of interest represent a pair of monomeric basolateral tubulo-vesicular structure localization signals that may be used together to target a molecule of interest to the membrane. The signals of this invention encompass localization signals linked to the N-terminus or C-terminus, or both the N-terminus and C-terminus of a polypeptide or other molecule of interest. SEQ ID NOS:5-8 are selected examples of subsequences of SEQ ID NOS:1-4, however, additional subsequences, wild type or mutated, may be utilized to form monomeric polarized cell tubulo-vesicular structure localization signals.
SEQ ID NOS:1-4 represent full length sequences of proteins that have tubulo-vesicular structure localization activity in polarized kidney epithelial cells. SEQ ID NOS:5-8 are subsequences of SEQ ID NOS:1-4 that are capable of conferring tubulo-vesicular structure localization. Polypeptide subsequences that are identical to their wild type parent may be used as part of a localization signal, however in one embodiment some amino acids are mutated to another amino acid, such as one of the naturally occurring amino acids including, alanine, aspartate, asparagine, cysteine, glutamate, glutamine, phenylalanine, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, arginine, valine, tryptophan, serine, threonine, or tyrosine. Mutation of amino acids may be performed for various reasons including, but not limited to, minimization of undesired biological activity, introduction or removal of secondary structure in the polypeptide; disruption of protein/protein interaction; modification of charge, hydrophobicity, or stability of the polypeptide; and introduction or removal of restriction sites in the nucleic acid encoding the polypeptide.
In general, tubulo-vesicular structure localization signals are built by identifying proteins that localize to the tubulo-vesicular structure and/or specific polarized locations of the tubulo-vesicular structure such as apical and basolateral locations. Sometimes it is desirable to utilize wild type truncations. However, it is sometimes desirable to modify one or more amino acids to enhance the localization. Other reasons for modifying the wild type sequences are to remove undesired characteristics, such as enzymatic activity or modulation of an endogenous cellular function. Monomeric signals may include a tubulo-vesicular structure localization sequence as well as amino acids adjacent and contiguous on either side. Monomeric signals may therefore be any length provided the monomer confers tubulo-vesicular structure localization. For example, the monomer may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30-100 or more amino acids adjacent to the tubulo-vesicular structure localization sequence.
For example, in one embodiment, the invention comprises a polypeptide containing an amino acid sequence at least 80% identical to SEQ ID NO:5 in frame with an amino acid sequence at least 80% identical to SEQ ID NO:6.
In another embodiment, the invention comprises a polypeptide containing an amino acid sequence at least 80% identical to SEQ ID NO:7 in frame with an amino acid sequence at least 80% identical to SEQ ID NO:8.
In another embodiment, the invention comprises a tubulo-vesicular structure localization signal comprising at least one copy of a peptide selected from the group consisting of:

- a) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 1-145 of the amino acid sequence of SEQ ID NO:1;
- b) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 1-140 of the amino acid sequence of SEQ ID NO:1;
- c) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 1-135 of the amino acid sequence of SEQ ID NO:1; and
- d) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 1-130 of the amino acid sequence of SEQ ID NO:1.

In another embodiment, the invention comprises a tubulo-vesicular structure localization signal comprising at least one copy of a peptide selected from the group consisting of:

- a) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 594-627 of the amino acid sequence of SEQ ID NO:2;
- b) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 591-627 of the amino acid sequence of SEQ ID NO:2;
- c) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 584-627 of the amino acid sequence of SEQ ID NO:2; and
- d) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 577-627 of the amino acid sequence of SEQ ID NO:2.

In another embodiment, the invention comprises a combination localization signal comprising a peptide at least 80% identical to a subsequence of SEQ ID NO:1 in frame with a peptide at least 80% identical to a subsequence of subsequence of SEQ ID NO:2.
In another embodiment, the invention comprises a localization signal comprising at least one copy of a peptide selected from the group consisting of:

- a) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 1-125 of the amino acid sequence of SEQ ID NO:3;
- b) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 1-130 of the amino acid sequence of SEQ ID NO:3;
- c) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 135 of the amino acid sequence of SEQ ID NO:3; and
- d) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 1-140 of the amino acid sequence of SEQ ID NO:3.

In another embodiment, the invention comprises a localization signal comprising at least one copy of a peptide selected from the group consisting of:

- a) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 581-602 of the amino acid sequence of SEQ ID NO:4;
- b) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 576-602 of the amino acid sequence of SEQ ID NO:4;
- c) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 573-602 of the amino acid sequence of SEQ ID NO:4; and
- d) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 5769-602 of the amino acid sequence of SEQ ID NO:4.

In another embodiment, the invention comprises a combination localization signal comprising a peptide at least 80% identical to a subsequence of SEQ ID NO:3 in frame with a peptide at least 80% identical to a subsequence of subsequence of SEQ ID NO:4.
In another embodiment, the invention comprises a tubulo-vesicular structure localization signal linked to an epitope.
In another embodiment, the invention comprises a tubulo-vesicular structure localization signal linked to a reporter.
In another embodiment, the invention comprises a tubulo-vesicular structure localization signal linked to a polypeptide of interest.
In another embodiment, the invention comprises a tubulo-vesicular structure localization signal linked to a therapeutic molecule.
In another embodiment, the invention comprises a nucleic acid molecule comprising a polynucleotide sequence encoding a localization signal.
In another embodiment, the invention comprises a vector comprising polynucleotide sequence encoding a localization signal.
In another embodiment, the invention comprises a recombinant host cell comprising the vector containing a polynucleotide sequence encoding a localization signal.
In another embodiment, the invention comprises a method of localizing a polypeptide of interest to a tubulo-vesicular structure subcellular compartment in a cell comprising linking a polypeptide of interest open reading frame to a nucleic acid molecule encoding a localization signal to create a fusion protein coding sequence, and transfecting the fusion protein coding sequence into a host cell and culturing the transfected host cell under conditions suitable to produce at least one copy of the fusion protein.
As used herein, the term “tubulo-vesicular structure” refers to describing novel subcellular structure(s) with characteristics similar to those in the figures (FIGS. 12-16) within this application.
As used herein, the terms “correspond(s) to” and “corresponding to,” as they relate to sequence alignment, are intended to mean enumerated positions within a reference protein, e.g., dopamine transporter (SEQ ID NO:1), and those positions that align with the positions on the reference protein. Thus, when the amino acid sequence of a subject peptide is aligned with the amino acid sequence of a reference peptide, e.g., SEQ ID NO:1, the amino acids in the subject peptide sequence that “correspond to” certain enumerated positions of the reference peptide sequence are those that align with these positions of the reference peptide sequence, but are not necessarily in these exact numerical positions of the reference sequence. Methods for aligning sequences for determining corresponding amino acids between sequences are described below.
Another embodiment of the invention is a nucleic acid molecule comprising a polynucleotide sequence encoding at least one copy of a polarized cell tubulo-vesicular structure localization signal polypeptide.
Another embodiment of the invention is a nucleic acid molecule comprising a polynucleotide sequence encoding at least two polarized cell tubulo-vesicular structure localization signal polypeptides.
Another embodiment of the invention is a nucleic acid molecule comprising a polynucleotide sequence encoding at least two different polarized cell tubulo-vesicular structure localization signal polypeptides.
Another embodiment of the invention is a nucleic acid molecule wherein the polynucleotide sequence encodes at least a number of copies of tubulo-vesicular structure localization signal selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9 or 10.
Another embodiment of the invention is a vector comprising a nucleic acid molecule encoding at least one copy of a polarized cell tubulo-vesicular structure localization signal.
Another embodiment of the invention is a recombinant host cell comprising a vector comprising a nucleic acid molecule encoding at least one copy of a tubulo-vesicular structure localization signal.
Another embodiment of the invention is a method of localizing a polypeptide to a tubulo-vesicular structure in a polarized cell comprising linking a polypeptide open reading frame to a localization signal open reading frame to create a fusion protein coding sequence, and transfecting the fusion protein coding sequence into a host cell and culturing the transfected host cell under conditions suitable to produce at least one copy of the fusion protein.
Another embodiment of the invention is a method of delivering a therapeutic molecule to a subcellular location in a cell comprising transfecting a vector comprising a nucleic acid molecule encoding at least one copy of a localization signal linked to a therapeutic molecule into a host cell and culturing the transfected host cell under conditions suitable to produce at least one copy of the localization signal-containing therapeutic molecule.
The invention also relates to modified localization signals that are at least about 80%, 85%, 90% 95%, 96%, 97%, 98% or 99% identical to a reference polypeptide. A modified localization signal is used to mean a peptide that can be created by addition, deletion or substitution of one or more amino acids in the primary structure (amino acid sequence) of a localization signal protein or polypeptide. The terms “protein” and “polypeptide” and “peptide” are used interchangeably herein. The reference polypeptide is considered to be the wild type protein or a portion thereof. Thus, the reference polypeptide may be a protein whose sequence was previously modified over a wild type protein. The reference polypeptide may or may not be the wild type protein from a particular organism.
A polypeptide having an amino acid sequence at least, for example, about 95% identical to a reference an amino acid sequence is understood to mean that the amino acid sequence of the polypeptide is identical to the reference sequence except that the amino acid sequence may include up to about five modifications per each 100 amino acids of the reference amino acid sequence encoding the reference peptide. In other words, to obtain a peptide having an amino acid sequence at least about 95% identical to a reference amino acid sequence, up to about 5% of the amino acid residues of the reference sequence may be deleted or substituted with another amino acid or a number of amino acids up to about 5% of the total amino acids in the reference sequence may be inserted into the reference sequence. These modifications of the reference sequence may occur at the N-terminus or C-terminus positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among amino acids in the reference sequence or in one or more contiguous groups within the reference sequence.
As used herein, “identity” is a measure of the identity of nucleotide sequences or amino acid sequences compared to a reference nucleotide or amino acid sequence. In general, the sequences are aligned so that the highest order match is obtained. “Identity” per se has an art-recognized meaning and can be calculated using published techniques. (See, e.g., Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York (1988); Biocomputing: Informatics And Genome Projects, Smith, D. W., ed., Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey (1994); von Heinje, G., Sequence Analysis In Molecular Biology, Academic Press (1987); and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York (1991)). While there exist several methods to measure identity between two polynucleotide or polypeptide sequences, the term “identity” is well known to skilled artisans (Carillo, H. & Lipton, D., Siam J Applied Math 48:1073 (1988)). Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego (1994) and Carillo, H. & Lipton, D., Siam J Applied Math 48:1073 (1988). Computer programs may also contain methods and algorithms that calculate identity and similarity. Examples of computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCG program package (Devereux, J., et al., Nucleic Acids Research 12(i):387 (1984)), BLASTP, ExPASy, BLASTN, FASTA (Altschul, S. F., et al., J Molec Biol 215:403 (1990)) and FASTDB. Examples of methods to determine identity and similarity are discussed in Michaels, G. and Garian, R., Current Protocols in Protein Science, Vol 1, John Wiley & Sons, Inc. (2000), which is incorporated by reference. In one embodiment of the present invention, the algorithm used to determine identity between two or more polypeptides is BLASTP.
In another embodiment of the present invention, the algorithm used to determine identity between two or more polypeptides is FASTDB, which is based upon the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990), incorporated by reference). In a FASTDB sequence alignment, the query and subject sequences are amino sequences. The result of sequence alignment is in percent identity. Parameters that may be used in a FASTDB alignment of amino acid sequences to calculate percent identity include, but are not limited to: Matrix=PAM, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the length of the subject amino sequence, whichever is shorter.
If the subject sequence is shorter or longer than the query sequence because of N-terminus or C-terminus additions or deletions, not because of internal additions or deletions, a manual correction can be made, because the FASTDB program does not account for N-terminus and C-terminus truncations or additions of the subject sequence when calculating percent identity. For subject sequences truncated at the N- and C-terminal ends, relative to the query sequence, the percent identity is corrected by calculating the number of amino acids of the query sequence that are N- and C-terminus to the reference sequence that are not matched/aligned, as a percent of the total amino acids of the query sequence. The results of the FASTDB sequence alignment determine matching/alignment. The alignment percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This corrected score can be used for the purposes of determining how alignments “correspond” to each other, as well as percentage identity. Residues of the query (subject) sequences or the reference sequence that extend past the N- or C-termini of the reference or subject sequence, respectively, may be considered for the purposes of manually adjusting the percent identity score. That is, residues that are not matched/aligned with the N- or C-termini of the comparison sequence may be counted when manually adjusting the percent identity score or alignment numbering.
For example, a 90 amino acid residue subject sequence is aligned with a 100 residue reference sequence to determine percent identity. The deletion occurs at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not show a match/alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C-termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%. In another example, a 90 residue subject sequence is compared with a 100 reference sequence. This time the deletions are internal deletions so there are no residues at the N- or C-termini of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected.
The localization signals of the invention optionally comprise spacer amino acids before or after them (for example, FIGS. 1D-1F, 2C-2F and 3D-3F). The length and composition of the spacer may vary. An example of a spacer is glycine, alanine, polyglycine, or polyalanine. In addition to providing space between peptide elements, spacers can be used for the purpose of engineering restriction sites in the encoding nucleic acid and can be used for modifying secondary structure of the polypeptide encoded. The spacer amino acids may be any amino acid and are not limited to alanine, glycine.
The localization signals of the invention are optionally linked to additional molecules or amino acids that provide an epitope, a reporter, and/or an experimental or therapeutic polypeptide or molecule (FIGS. 1A-1F, 2A-2G and 3A-3F). Non-limiting examples of epitopes are FLAG™ (Kodak; Rochester, N.Y.), HA (hemaglutinin), c-Myc and His6. Non-limiting examples of reporters are alkaline phosphatase, galactosidase, peroxidase, luciferase and fluorescent proteins. Non-limiting examples of experimental proteins or polypeptides of interest are enzymes, enzyme binding partners, signalling factors, structural factors, and peptide ligands, metabolic binding factors, nucleic acid binding factors, and cellular binding factors. The epitopes, reporters and experimental or therapeutic molecules are given by way of example and without limitation. The epitope, reporter, experimental molecule and/or therapeutic molecule may be the same molecule. The epitope, reporter, experimental molecule and/or therapeutic molecule may also be different molecules.
Localization signals and optional amino acids linked thereto can be synthesized chemically or recombinantly using techniques known in the art. Chemical synthesis techniques include but are not limited to peptide synthesis which is often performed using an automated peptide synthesizer. Peptides can also be synthesized utilizing non-automated peptide synthesis methods known in the art. Recombinant techniques include insertion of localization signal encoding nucleic acids into expression vectors, wherein nucleic acid expression products are synthesized using cellular factors and processes.
Linkage of an epitope, reporter, experimental or therapeutic molecule to a localization signal can include covalent or enzymatic linkage. When the localization signal comprises material other than a polypeptide, such as a lipid or carbohydrate, a chemical reaction to link molecules may be utilized. Additionally, non-standard amino acids and amino acids modified with lipids, carbohydrates, phosphate or other molecules may be used as precursors to peptide synthesis.
The localization signals of the invention have utility as therapeutic targeting molecules. Polypeptides or proteins of interest linked to localization signals have utility as therapeutics or as subcellular tools. For example, the fusion polypeptides depicted generically in FIGS. 1A-1F and 2A-2G have utility as subcellular tools or therapeutics. Localization signal-containing gene constructs are also delivered via gene therapy. FIGS. 5B and 5C depict embodiments of gene therapy vectors for delivering and controlling polypeptide expression in vivo. Polynucleotide sequences linked to the gene construct in FIGS. 5B and 5C include genome integration domains to facilitate integration of the transgene into a viral genome and/or host genome.
FIG. 5A shows a vector containing a tubulo-vesicular structure localization signal, optional epitope, and protein of interest gene construct, wherein the gene construct is releasable from the vector as a unit useful for generating transgenic animals. For example, the gene construct, or transgene, is released from the vector backbone by restriction endonuclease digestion. The released transgene is then injected into pronuclei of fertilized mouse eggs; or the transgene is used to transform embryonic stem cells. The vector containing a localization signal and reporter gene construct of FIG. 5A is also useful for transient transfection of the transgene, wherein the promoter and codons of the transgene are optimized for the host organism. The vector containing a gene construct of FIG. 5A is also useful for recombinant expression of polypeptides in fermentable organisms adaptable for small or large scale production, wherein the promoter and codons of the transgene are optimized for the fermentation host organism.
FIG. 5D shows a vector containing a polarized cell tubulo-vesicular structure localization signal gene construct useful for generating stable cell lines.
The invention also encompasses polynucleotides comprising nucleotide sequences encoding polarized cell tubulo-vesicular structure localization signals. The polynucleotides of the invention are optionally linked to additional nucleotide sequences encoding epitopes, reporters and/or experimental or therapeutic molecules. Further, the nucleic acids of the invention are optionally incorporated into vector polynucleotides. The polynucleotides are optionally flanked by nucleotide sequences comprising restriction endonuclease sites and other nucleotides needed for restriction endonuclease activity. The flanking sequences optionally provide cloning sites within a vector. The restriction sites can include, but are not limited to, any of the commonly used sites in most commercially available cloning vectors. Non-limiting examples of such sites are those recognized by NsiI, ApaLI, MfeI, KpnI, BamHI, ClaI, EcoRI, EcoRV, SpeI, AflII, NdeI, NheI, XbaI, XhoI, SphI, NaeI, SexAI, HindIII, HpaI, and PstI restriction endonucleases. Sites for cleavage by other restriction enzymes, including homing endonucleases, are also used for this purpose. The polynucleotide flanking sequences also optionally provide directionality of subsequence cloning. It is preferred that 5′ and 3′ restriction endonuclease sites differ from each other so that double-stranded DNA can be directionally cloned into corresponding complementary sites of a cloning vector.
Localization signals with or without epitopes, reporters, or experimental or therapeutic proteins are alternatively synthesized by recombinant techniques. Polynucleotide expression constructs are made containing desired components and inserted into an expression vector. The expression vector is then transfected into cells and the polypeptide products are expressed and isolated. Localization signals made according to recombinant DNA techniques have utility as research tools and/or subcellular therapeutic delivery agents.
The following is an example of how polynucleotides encoding localization signals are produced. Complimentary oligonucleotides encoding the localization signals and flanking sequences are synthesized and annealed. The resulting double-stranded DNA molecule is inserted into a cloning vector using techniques known in the art. When the localization signals are placed in-frame adjacent to sequences within a transgenic gene construct that is translated into a protein product, they form part of a fusion protein when expressed in cells or transgenic animals.
Another embodiment of the invention relates to selective control of transgene expression in a desired cell or organism. The promoter portion of the recombinant gene can be a constitutive promoter, a non-constitutive promoter, a tissue-specific promoter (constitutive or non-constitutive) or a selectively controlled promoter. Different selectively controlled promoters are controlled by different mechanisms. RheoSwitch® is an inducible promoter system available from New England Biolabs (Ipswich, Mass.) (FIGS. 8-11). Temperature sensitive promoters can also be used to increase or decrease gene expression. An embodiment of the invention comprises a localization signal-containing gene construct whose expression is controlled by an inducible or repressible promoter. In one embodiment, the inducible promoter is tetracycline controllable.
Polarized cell tubulo-vesicular structure localization signals and methods of making these localization signals are disclosed. Below are examples of methods of using tubulo-vesicular structure localization signals. In general, localization signals linked to epitopes, reporters, and other desired proteins or molecules are delivered via adenovirus, lentivirus, adeno-associated virus, or other viral constructs that express protein product in a cell.

Example 1

A transgene is constructed using a human cytomegalovirus (CMV) promoter to direct expression of a fusion protein comprising SEQ ID NO:7 (LOCALIZATION SIGNAL) and green fluorescent protein (REPORTER). Such a transgene is generically represented by FIG. 4G. The transgene is transfected into cells for transient expression. Verification of expression and location is performed by visualization of the fluorescent protein by confocal microscopy.

Example 2

A transgene is constructed using a human cytomegalovirus (CMV) promoter to direct expression of a fusion protein comprising green fluorescent protein (REPORTER) and SEQ ID NO:8 (LOCALIZATION SIGNAL). Such a transgene is generically represented by FIG. 4H. The transgene is transfected into cells for transient expression. Verification of expression and location is performed by visualization of the fluorescent protein by confocal microscopy.

Example 3

A transgene is constructed using a human cytomegalovirus (CMV) promoter to direct expression of a fusion protein comprising SEQ ID NO:7 (N-terminal LOCALIZATION SIGNAL 1), green fluorescent protein (REPORTER), and SEQ ID NO:8 (C-terminal LOCALIZATION SIGNAL 2). Such a transgene is generically represented by FIG. 4C. The transgene is transfected into cells for transient expression. Verification of expression and location is performed by visualization of the fluorescent protein by confocal microscopy.

Example 4

A transgene is constructed using a human cytomegalovirus (CMV) promoter to direct expression of a fusion protein comprising SEQ ID NO:5 (LOCALIZATION SIGNAL) and green fluorescent protein (REPORTER). Such a transgene is generically represented by FIG. 4G. The transgene is transfected into cells for transient expression. Verification of expression and location is performed by visualization of the fluorescent protein by confocal microscopy.

Example 5

A transgene is constructed using a human cytomegalovirus (CMV) promoter to direct expression of a fusion protein comprising green fluorescent protein (REPORTER) and SEQ ID NO:6 (LOCALIZATION SIGNAL). Such a transgene is generically represented by FIG. 4H. The transgene is transfected into cells for transient expression. Verification of expression and location is performed by visualization of the fluorescent protein by confocal microscopy.

Example 6

A transgene is constructed using a human cytomegalovirus (CMV) promoter to direct expression of a fusion protein comprising SEQ ID NO:5 (N-terminal LOCALIZATION SIGNAL 1), green fluorescent protein (REPORTER), and SEQ ID NO:6 (C-terminal LOCALIZATION SIGNAL 2). Such a transgene is generically represented by FIG. 4C. The transgene is transfected into cells for transient expression. Verification of expression and location is performed by visualization of the fluorescent protein by confocal microscopy.

Example 7

A transgene construct is built to produce a protein product with expression driven by a tissue-specific promoter. The transgene comprises a synthetic gene expression unit engineered to encode three domains. Each of these three domains is synthesized as a pair of complimentary polynucleotides that are annealed in solution, ligated and inserted into a vector. Starting at the amino-terminus, the three domains in the expression unit are nucleotide sequences that encode a kinase inhibitor, a FLAG™ epitope, and a basolateral tubulo-vesicular structure localization signal (for example, SEQ ID NO:8). Nucleotide sequences encoding a FLAG™ epitope are placed downstream of nucleotide sequences encoding the kinase inhibitor. Finally, nucleotide sequences encoding the localization signal are placed downstream of those encoding the FLAG™ epitope. The assembled gene expression unit is subsequently subcloned into an expression vector, such as that shown in FIG. 5A, and used to transiently transfect cells polarized. Verification is performed by microscopic visualization of the epitope immunoreactivity at the basolateral tubulo-vesicular structure of polarized cells.

Example 8

Localization of a polypeptide of interest is demonstrated in vivo by making a transgene construct used to generate mice expressing a fusion protein targeted to the tubulo-vesicular structure of kidney epithelial cells. The transgene construct is shown generically in FIG. 4C. The expression unit contains nucleotides that encode SEQ ID NO:5 (N-terminal LOCALIZATION SIGNAL 1), polypeptide of interest (POLYPEPTIDE OF INTEREST), and SEQ ID NO:6 (C-terminal LOCALIZATION SIGNAL 2). This expression unit is subsequently subcloned into a vector between nucleotide sequences including a kidney epithelial cell-specific promoter and an SV40 polyadenylation signal. The completed transgene is then injected into pronuclei of fertilized mouse oocytes. The resultant pups are screened for the presence of the transgene by PCR. Transgenic founder mice are bred with wild-type mice. Heterozygous transgenic animals from at least the third generation are used for the following tests, with their non-transgenic littermates serving as controls.
Test 1: Southern blotting analysis is performed to determine the copy number. Southern blots are hybridized with a radio-labeled probe generated from a fragment of the transgene. The probe detects bands containing DNA from transgenic mice, but does not detect bands containing DNA from non-transgenic mice. Intensities of the transgenic mice bands are measured and compared with the transgene plasmid control bands to estimate copy number. This demonstrates that mice in Example 8 harbor the transgene in their genomes.
Test 2: Kidney cells are prepared for microscopic analysis. Visualization is performed using a fluorescently labeled antibody against the polypeptide of interest. This experiment demonstrates the transgene is expressed at the tubulo-vesicular structure of kidney epithelial cells in transgenic mice because the polypeptide of interest is visualized in transgenic kidney cells but not in non-transgenic tissues.
These examples demonstrate delivery of a polypeptide or other molecule of interest to a localized region of a cell for therapeutic or experimental purposes. Nucleotide sequences encoding the localization signals permit incorporation into a vector designed to deliver and express a gene product in a cell or organism. Such vectors include plasmids, cosmids, artificial chromosomes, and modified viruses. Delivery to eukaryotic cells can be accomplished in vivo or ex vivo. Ex vivo delivery methods include isolation of the intended recipient's cells or donor cells and delivery of the vector to those cells, followed by treatment of the recipient with the cells.

Example 9

The transgene constructs harboring tubulo-vesicular structure localization signal are expressed and localized to the basal surface of mammalian cells (FIGS. 12-16). To demonstrate the tubulo-vesicular structure localization, the mammalian cell line MDCK (ATCC #CCL-34), canine normal kidney cells, was used. Cell were plated on poly-L-lysine coated MatTek Cultureware 24 well glass bottom plates (#P24G-1.5-13-F) at a density of 15,000 cells/cm². The cells were then transfected using Fugene6 transfection reagent (Roche) at a ratio of 1.2 μl Fugene6 reagent to 0.4 μg of total DNA per well. The ratio of intergrase vector to specific vector was 3:1; 0.3 μg of VVN-3217 (FIG. 18) to 0.1 μg of VVN859x (FIG. 19). The transfected cells were grown at 37° C. for 3 days before induction with RSL1 at 100 nM. The cells were induced for 18 hr before fixation with 4% paraformaldehyde for 10 min. The cells were washed 3 times with PBS for 5 min per wash. Blocking was performed for 30 minutes using 5% bovine serum albumin in PBS (BSA/PBS) at room temperature followed by washing the cells as above. Primary antibodies were diluted in 1% BSA/PBS at a dilution of 1:100. VVN-8595 (FIGS. 8 and 12) and VVN-8596 (FIGS. 9 and 13) were stained with mouse-anti-FLAG (Sigma) and VVN-8598 (FIGS. 10 and 14) and VVN-8599 (FIGS. 11, 15 and 16) were stained with rat-anti-HA (Roche) for 1 hour at room temperature. After the cells were washed as above and secondary antibodies were added at 1:1000 diluted in 1% BSA/PBS for 1 hour. VVN-8595 (FIGS. 8 and 12) and VVN-8596 (FIGS. 9 and 13) were stained with goat-anti-mouse Alexa488 and VVN-8598 (FIGS. 10 and 14) and VVN-8599 (FIGS. 11, 15 and 16) were stained with rabbit-anti-rat Alexa488. The cells were washed as above and stained with DAPI to stain the nucleus of the MDCK cells for 5 minutes followed by washing as above. The cells were then imaged on the Zeiss Observer in a Z-stack at 63×.
Disclosed are polarized cell tubulo-vesicular structure localization signals and methods of making and using these localization signals. The localization signals are utilized as research tools or as therapeutic delivery agents. The invention includes linking polypeptides or other molecules of interest to cellular localization signals for subcellular therapeutics.

Claims

1. A polypeptide containing an amino acid sequence at least 80% identical to SEQ ID NO:5 in frame with an amino acid sequence at least 80% identical to SEQ ID NO:6.

2. A polypeptide containing an amino acid sequence at least 80% identical to SEQ ID NO:7 in frame with an amino acid sequence at least 80% identical to SEQ ID NO:8.

3. A combination localization signal comprising a peptide at least 80% identical to a subsequence of SEQ ID NO:1 in frame with a peptide at least 80% identical to a subsequence of subsequence of SEQ ID NO:2.

4. A combination localization signal comprising a peptide at least 80% identical to a subsequence of SEQ ID NO:3 in frame with a peptide at least 80% identical to a subsequence of subsequence of SEQ ID NO:4.

5. A tubulo-vesicular structure localization signal comprising at least one copy of a peptide selected from the group consisting of:

a) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 1-145 of the amino acid sequence of SEQ ID NO:1;

b) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 1-140 of the amino acid sequence of SEQ ID NO:1;

c) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 1-135 of the amino acid sequence of SEQ ID NO:1; and

d) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 1-130 of the amino acid sequence of SEQ ID NO:1.

6. A localization signal comprising at least one copy of a peptide selected from the group consisting of:

a) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 594-627 of the amino acid sequence of SEQ ID NO:2;

b) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 591-627 of the amino acid sequence of SEQ ID NO:2;

c) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 584-627 of the amino acid sequence of SEQ ID NO:2; and

d) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 577-627 of the amino acid sequence of SEQ ID NO:2.

7. A combination localization signal comprising a peptide of claim 5 in frame with a peptide of claim 6.

8. A localization signal comprising at least one copy of a peptide selected from the group consisting of:

a) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 1-125 of the amino acid sequence of SEQ ID NO:3;

b) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 1-130 of the amino acid sequence of SEQ ID NO:3;

c) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 135 of the amino acid sequence of SEQ ID NO:3; and

d) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 1-140 of the amino acid sequence of SEQ ID NO:3.

9. A localization signal comprising at least one copy of a peptide selected from the group consisting of:

a) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 581-602 of the amino acid sequence of SEQ ID NO:4;

b) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 576-602 of the amino acid sequence of SEQ ID NO:4;

c) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 573-602 of the amino acid sequence of SEQ ID NO:4; and

d) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 5769-602 of the amino acid sequence of SEQ ID NO:4.

10. A combination localization signal comprising a peptide of claim 8 in frame with a peptide of claim 9.

11. The localization signal of claim 3 linked to an epitope.

12. The localization signal of claim 3 linked to a reporter.

13. The localization signal of claim 3 linked to a polypeptide of interest.

14. The localization signal of claim 3 linked to a therapeutic molecule.

15. A nucleic acid molecule comprising a polynucleotide sequence encoding at least one copy of the peptide of claim 1.

16. A vector comprising the nucleic acid molecule of claim 15.

17. A recombinant host cell comprising the vector of claim 16.

18. A method of localizing a polypeptide of interest to a tubulo-vesicular structure subcellular compartment in a cell comprising linking a polypeptide of interest open reading frame to a nucleic acid molecule of claim 15 to create a fusion protein coding sequence, and transfecting the fusion protein coding sequence into a host cell and culturing the transfected host cell under conditions suitable to produce at least one copy of the fusion protein.