WO2009129619A1

WO2009129619A1 - Aptamers for detection and imaging of apoptosis

Info

Publication number: WO2009129619A1
Application number: PCT/CA2009/000533
Authority: WO
Inventors: John Mercer; Janice Kapty; David Murray; Chris X Le; Alex Ebhardt; Richard Fahlman
Original assignee: Alberta Cancer Board
Current assignee: Alberta Cancer Board
Priority date: 2008-04-22
Filing date: 2009-04-22
Publication date: 2009-10-29
Anticipated expiration: 2010-10-22

Abstract

The invention is directed to labeled phosphatidylserine-specific aptamers for use in methods of detection and imaging of apoptotic cells.

Description

3409-255

April 22, 2009

Filing Version

Aptamers for Detection and Imaging of Apoptosis

Field of the Invention

The present invention relates to the detection of cell death. More specifically, the invention relates to aptamers as well as methods for the generation and use of aptamers that selectively bind to cells undergoing apoptosis as agents for the detection, monitoring and imaging of apoptotic cells such as cancer cells.

Background of the Invention

For years work has been carried out on the identification of molecules characteristic and unique in tumours, or molecules altered, over expressed or otherwise differing in cancer. These molecules are known as tumour markers and have been used over the years as the target for many immunological approaches to cancer imaging, diagnosis and therapy.

Cancer therapies typically involve the ablation of tumour cells and tissues. For patients undergoing therapy for cancer there are highly effective treatments and treatment options, however, it is often difficult to choose the most appropriate treatment since it takes some time before a tumour will show signs of effective therapy. There are some very rapid changes that occur at the cellular level that would be useful to use to provide a quick assessment of the success of a particular treatment option. When treatment is effective and cancer cells are destined to die they undergo a programmed cell death, a process called apoptosis. The process of apoptosis involves a cascade of cytoplasmic and nuclear events that result in a series of morphological changes and eventually cause the demise of the cell. Apoptosis is characterized by distinct biochemical and morphological changes exhibited by cells undergoing programmed cell death, including DNA fragmentation, plasma membrane blebbing and cell volume shrinkage. In particular, when cells undergo apoptosis, a phospholipid called phosphatidylserine (PS), is externalized to the outer lipid membrane layer. In normal cells PS is restricted to the inner layer of the membrane.

Researchers have developed strategies to be able to image apoptotic events in cells. However, the research is typically directed to the use of peptides or proteins as the indicators of apoptosis. Enzymatic targets to detect apoptosis are disclosed for example in U.S. 5,976,822, U.S. 6,596,480 and U.S. 7,270,801. Antibody approaches to detect apoptotic cells are known and disclosed for example in U.S. 7,037,664. AnnexinV is a protein that has also been developed as a target for apoptotic cells. AnnexinV possesses a high affinity for anionic phospholipid surfaces, such as a membrane leaflet having an exposed surface of phosphatidylserine (PS). U.S. 6,726,895 discloses the use of radiolabeled AnnexinV for imaging apoptotic cells. U.S. 6,843,980 discloses methods for using AnnexinV for detecting apoptotic cells. However, there are drawbacks to using AnnexinV as a target molecule for apoptosis. For example, its use requires a fairly high concentration of calcium for activity. Furthermore, labeled AnnexinV does not target apoptotic cells with sufficient specificity and availability and thus a marker with better imaging capabilities is desirable. U.S. 6,949,350 discloses methods for determining chemosensitivity of cells towards a substance by measuring apoptosis induced by the substance. General protein markers that interact with phophatidylserine are generally described , however, only AnnexinV is demonstrated.

Aptamers are DNA or RNA molecules that have been selected from random pools based on their ability to bind other molecules and thus can be used as targeting moieties. Aptamers have been selected which bind nucleic acid, proteins, small organic compounds, and even cells. Methods and compositions for identifying and making aptamers are known to those of skill in the art and are described e.g., in U.S. 5,840,867 and U.S. 5,582,981 (the disclosures of which are each incorporated herein by reference in their entirety). Created by an in vitro selection process from pools of random sequence oligonucleotides, aptamers have been generated for over 100 proteins including growth factors, transcription factors, enzymes, immunoglobulins, and receptors. A typical aptamer is 10-15 kDa in size (15-90 nucleotides), binds its target with sub-nanomolar affinity, and discriminates against closely related targets (e.g., aptamers will typically not bind other proteins from the same gene family).

Aptamers have a number of desirable characteristics for use as therapeutics and diagnostics including high specificity and affinity, biological efficacy, and excellent pharmacokinetic properties. In addition, they offer specific competitive advantages over antibodies and other protein biologies: they can be produced by an entirely in vitro process; they have little or no toxicity or immunogenicity compared to antibodies; can be administered by subcutaneous injection ; are chemically robust and stable; have a small size allowing them to penetrate into areas of conformational constrictions that do not allow for antibodies or antibody fragments.

There is currently no known aptamer or nucleic acid agent that specifically targets phospholipids to detect apoptotic cells. Given the advantages of aptamer technology it would be useful to develop aptamers that could recognize and target externalized phosphatidylserine in apoptotic cells. This would be advantageous over prior art methods of targeting apoptotic cells. The present invention provides novel methods to produce aptamers that detect apoptotic cells via recognition of phosphatidylserine (PS). The novel aptamers can be used in a variety of novel methods to monitor the effectiveness of cancer therapies in subjects.

Summary of the Invention

The present invention provides novel aptamers that specifically target phosphatidylserine (PS) on the cell surface of cells undergoing apoptosis. The present invention also provides novel methods of generating such aptamers. The aptamers of the invention are used in methods to image cells undergoing apoptosis. As such the invention also provides uses of the aptamers for the monitoring of apoptotic cells to determine efficacy of treatments used in cancer patients. The novel aptamers and methods can be used in vitro, ex vivo and in vivo. According to an aspect of the present invention is an aptamer that selectively binds to a phospholipid. In aspects the phospholipid is phosphatidylserine (PS).

According to another aspect of the invention is a phosphatidylserine specific aptamer.

According to another aspect of the invention is a labeled phosphatidylserine specific aptamer.

According to another aspect of the invention is a labeled phosphatidylserine specific oligonucleotide aptamer. In aspects the oligonucleotide labeled PS-specific aptamer is about 15 to about 90 nucleotides in length. In further aspects the oligonucleide labeled PS-specific aptamer is about 1-15 kDa.

According to a yet further aspect of the present invention, there is provided an aptamer or composition comprising the aptamer of the invention for use in imaging.

According to yet another aspect of the invention, there is provided an aptamer or composition comprising the aptamer of the invention for use in diagnosis.

According to yet another aspect of the invention, there is provided an aptamer or composition comprising the aptamer of the invention for use in screening of pharmaceuticals.

According to another aspect of the present invention, there is provided a method of imaging a disease or condition involving apoptosis, the method comprising the step of administering an aptamer or pharmaceutical composition comprising said aptamer to a sample or subject, where the aptamer selectively binds to cells undergoing apoptosis.

According to another aspect of the invention is a method for targeting or imaging a tissue in a subject comprising administering to the subject an aptamer comprising a plurality of targeting/imaging molecules covalently attached to said aptamer, and delivering said aptamer to tissue in the subject, wherein said aptamer is selective for phosphatidylserine (PS) and wherein binding of said aptamer to said tissue is an indication of apoptotic cells in said tissue.

A screening method for the identification of agents which modulate, either directly or indirectly, apoptosis of a cell, the method comprising, i) providing at least one candidate agent to be tested to a population of cells; ii) providing a phosphatidylserine specific labeled aptamer to said population of cells, and iii) determining the effect, or not, of said agent on apoptosis of said cells.

According to another aspect of the invention is a liposome containing phosphatidylserine (herein referred to as "PS-L"). In aspects, the PS-L liposome is used in methods to produce PS-targeting aptamers. In further aspects, the procedure is SELEX™.

According to another aspect of the invention is an imaging composition comprising an imaging probe to a PS-specific aptamer having an imaging probe attached thereto, and an aqueous vehicle.

According to yet another aspect of the present invention is a method for determining the effectiveness of the treatment of cancerous cells and/or tissues, the method comprising administering a labeled PS-specific aptamer to said cells and/or tissues, wherein binding of said labeled PS-specific aptamer can be detected and is an indication that said cancerous cells and/or tissues are undergoing apoptosis and said treatment is effective.

According to yet another aspect of the present invention is an in vivo method for determining the effectiveness of a cancer treatment in a subject, the method comprising administering a labeled PS-specific aptamer to said subject undergoing a cancer treatment, wherein binding of said labeled PS-specific aptamer is detected and is an indication that said treatment is effective.

According to another aspect of the present invention is a method for producing a PS specific aptamer to detect apoptotic cells, the method comprising:

(a) preparing a nucleic acid test mixture;

(b) coupling each member of said nucleic acid test mixture with a first reactant consisting of a PS-liposome to form a nucleic acid-first reactant test mixture;

(c) forming a product library by contacting said nucleic acid-first reactant test mixture with a mixture of free reactants, wherein said product library is formed as a result of a bond formation reaction between said first reactant and at least one of said free reactants, wherein said bond formation reaction is facilitated by a nucleic acid coupled to said first reactant;

^d) -contacting the product library of step (c) with a target, wherein the product having the ability to perform a preselected function on said target relative to the product library may be partitioned from the remainder of the product library; and

(e) partitioning said product having said ability to perform a preselected function on said target from the remainder of the product library, whereby said product is identified. According to a further aspect of the invention, there is provided a method for generating DNA or RNA aptamers specific to phosphatidylserine (PS), the method comprising:

(a) contacting a mixture of randomly generated nucleic acid sequences of fixed length flanked by constant 5[ and 3^ ends with target molecules (PS liposomes) under conditions favorable for binding;

(b) partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules;

(c) dissociating the nucleic acid-target complexes;

(d) amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids; and

(e) reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific, high affinity nucleic acid ligands to the target molecule.

Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from said detailed description.

Brief Description of the Drawings

The present invention will become more fully understood from the detailed description given herein and from the accompanying drawings, which are given by way of illustration only and do not limit the intended scope of the invention. Figure 1 is a schematic diagram of the SELEX™ procedure using PS coated liposomes to generate PS-specific aptamers in one embodiment of the invention.

Figure 2 is a gel indicating PCR amplification of DNA library. Lane 1: 100 bp marker, lane 2: single stranded DNA library, lanes 3-5: cycles 1-3.

Figure 3 is a gel image of PCR amplified DNA pool from Round 1. Lane on far left is DNA ladder. Lane 2 is 10 cycles, lane 3 is 13 cycles and lane 4 is 15 cycles of PCR amplification.

Figure 4 is a gel image of PCR amplified DNA pool from Round 2. Lane 1 (far left) is DNA ladder. Lane 2 is the material out of Round 2 after 12 cycles of PCR amplification.

Figure 5 is a gel image of PCR amplified DNA pool from Round 3. Two lanes on left are DNA ladder. The numbers 15, 18, 20, and 22 indicate the number of cycles in the PCR reaction.

Figure 6 is an agarose gel of PCR amplified DNA pool from Round 3R, where DNA is stained with ethidium bromide. Lane 1: 15 cycles of PCR, lane 2: 18 cycles, lane 3: 20 cycles and lane 4: 22 cycles (far right lane).

Figure 7 shows PCR amplified DNA pool from Round 4R. The left lane is the DNA ladder. The numbers 15 and 19 indicate the number of cycles in the PCR reaction. Lane 2/3 are supernatant. Lane 4/5 are wash 1. Lanes 6/7 are the DNA bound to the pellet.

Figure 8 shows PCR amplified DNA pool from Round 5R. Lane 1 (on left) is DNA ladder. Lane 2/3 is supernatant with 15 and 20 cycles, respectively. Lane 4/5 is wash 1 with 15 and 20 cycles, respectively. Lane 6/7 are DBA bound to the pellet 15 and 20 cycles of PCR amplification, respectively. Figure 9 shows PCR amplified DNA pool from Round 6R. Lane 1 (on left) is DNA ladder. Lane 2/3 is PCR amplified water. Lane 4/5 is supernatant. Lane 6/7 are wash 1. Lane 9/10 are DNA bound to the pellet. The numbers 15 and 20 indicate the number of cycles in the PCR reaction.

Figure 10 shows PCR amplified DNA pool from Round 7R. Lane 1 is supernatant after 15 cycles of PCR. Lane 2 is supernatant after 20 cycles of PCR. Lane 3 is Wash 1 after 15 cycles of PCR. Lane 4 is Wash 1 after 20 cycles of PCR. Lane 5 is Wash 2 after 15 cycles of PCR. Lane 6 is Wash 2 after 20 cycles of PCR. Lane 7 is Wash 3 after 15 cycles of PCR. Lane 8 is Wash 3 after 20 cycles of PCR. Lane 9 is pellet DNA after 15 cycles of PCR. Lane 10 is pellet DNA after 20 cycles of PCR.

Figure 11 shows PCR amplified DNA pool from Round 8R. Lane 1 is DNA ladder. Lane 2/3 is supernatant. Lane 4/5 is wash 1. Lane 6/7 is Wash 2. Lane 8/9 are DNA bound to the pellet, with the left lane of each pair representing 15 cycles of PCR amplification, while the right lane of each pair represents 20 cycles of PCR.

Figure 12 shows PCR amplified DNA from Round 9R. Lane 1 is RO supernatant. Lane 2 is RO wash 1. Lane 3 is RO bound to pellet. Lane 4 is empty. Lane 5 is R9 supernatant. Lane 6 is R9 wash 1. Lane 7 is R9 bound to pellet.

Figure 13 shows a graph comparing binding of pool RO and R9R to liposomes containing PS which demonstrates the aptamers produced by the methods of the invention do bind to PS.

Detailed Description of the Embodiments

The present invention provides methods and compositions for imaging apoptotic cells and tissues both in vitro and in vivo. The present invention is based, at least in part, on the development of novel aptamers that are specific for binding to phosphatidylserine (PS) on apoptotic cells. Thus the aptamers of the invention allow for the efficient and effective detection of cells undergoing cell death. The present invention is also based on the combination of the PS-specific aptamer with an imaging probe allowing for the efficient and effective detection of apoptotic cells and tissues by various imaging methods.

The present invention is based on the generation of aptamers that are specific to target phosphatidylserine (PS) that is found on the outside of cells undergoing apoptosis. Using liposome technology, PS containing liposomes were produced that mimic the surface of cells undergoing apoptosis. In particular the liposomes generated have a percentage of the external membrane surface composed of phosphatidylserine. This PS liposome was then used in combination with a SELEX™ selection process to produce and isolate PS-specific aptamers that bind to cells undergoing apoptosis.

To the PS-specific aptamers can be attached a selected imaging probe (i.e. herein referred to as a labeled PS-specific aptamer). Imaging probes can be those that emit radioactive particles or electromagnetic emissions such as gamma rays, x-rays, light or ultraviolet or infrared photons and are known to those of skill in the art. Imaging probes can also be chemical or enzymatic in nature. The labeled PS-specific aptamer can be used in vitro, ex vivo and in vivo to detect cells undergoing apoptosis. The detection can be done on cell culture, tissue culture and cell and tissue biopsy material. The detection can be done non-invasively in patients undergoing cancer treatment. This provides a novel method for the efficient and effective detection of apoptotic cells and tissues in vivo by various imaging methods in those conditions involving apoptosis. This will provide an indication of the effectiveness of the cancer treatment being administered to a subject. This effectiveness can be done minutes to hours to days to weeks to months after the initiation of the cancer treatment and during cancer treatment in a subject.

The present invention also involves methods of making liposomes containing sufficient amounts of phosphatidylserine to use in method to generate PS-specific aptamers. In aspects, the liposomes may contain varying amounts of PS such as for example about 1% to about 40%. In some aspects up to about 40% PS, up to about 30% PS, up to about 20%, up to about 15%, up to about 10%, up to about 5% and any amounts in any of the ranges provided herein.

In one embodiment the condition involving apoptosis is cancer. Cancer treatments/therapies are directed to the killing of the cancerous cells and tissues. Any type of cancer is embodied within the present invention. As there are many available cancer treatments it is difficult to select the most effective treatment for each subject since it takes some time before a tumour will show sign of effective therapy. In accordance with the present invention, the labeled PS-specific aptamer can be provided as an aqueous composition and administered to a subject to detect apoptosis of the cells/tissues as a sign of the effectiveness of the treatment. This will allow the physician to assess the effectiveness of cancer chemotherapy or radiotherapy in conjunction with the treatment. Thus the present invention provides a quick measure of the ability of a particular therapy to kill cells in the tumour. Since the process of apoptosis is initiated very rapidly and since the cell surface expression of phosphatidylserine is one of the earliest manisfestations of apoptosis, the developed labeled PS-specific aptamer of the invention can be used within hours of the initiation of therapy to provide evidence of a therapeutic response.

Thus in embodiments of the invention the PS-specific aptamers can be generally be used to detect apoptosis or increases or decreases in apoptosis in any condition or treatment. In some aspects, the labeled PS-specific aptamers of the invention are useful for detecting treatment-induced (targeted) increases in apoptosis in conditions characterized by reduced apoptosis. Non-limiting examples of such conditions include neoplastic diseases, such as cancerous and benign tumours; autoimmune diseases, such as systemic lupus erythematosus, and myasthenia gravis; inflammatory diseases, such as bronchial asthma, inflammatory intestinal disease, and pulmonary inflammation; and viral infections, such as adenovirus, baculovirus, and poxvirus. In other aspects, the labeled PS-specific aptamers are useful for detecting treatment-induced decreases in apoptosis in conditions characterized by increased apoptosis. Non-limiting examples of such conditions include neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, and epilepsy; hematologic diseases, such as aplastic anemia, myelodysplastic syndrome, T CD4⁺ lymphocytopenia, and G6PD deficiency; and conditions resulting from tissue damage, such as myocardial infarction, cerebrovascular trauma, ischemic renal damage, and polycycstic kidney. The PS-specific aptamers are also useful for detecting apoptotic cells associated with the ageing process and any changes in cell death associated with treating the signs of ageing.

In a non-limiting embodiment, the labeled PS-specific aptamer of the invention would be formulated as an aqueous composition and administered by intravenous injection a few minutes and/or hours after the initiation of a course of a desired therapy such as for example chemotherapy. After a short time of minutes to hours the cancer subject would be imaged using a nuclear medicine procedure and cameras designed to provide pictures of the localized radioactivity within the body. If the cancer tissue is responding to the chemotherapy it would be targeted by the circulating radioactively labeled PS-specific aptamer and the camera would indicate increased radioactivity in the cancer tissue relative to normal tissues. Thus within several hours the suitability of the chosen chemotherapy could be determined.

In further embodiments of the invention the labeled PS-specific aptamer can be used in fundamental research in cell culture and animal models of cancer.

I. Definitions

As used herein, the term "apoptosis" refers to programmed cell death whereby the cell executes a cell suicide program.

As used herein, the term "subject" includes warm-blooded animals, preferably mammals, including humans. In a preferred embodiment, the subject is a human. Apoptosis may be imaged or detected in, for example, an organ of a subject or a portion thereof (e.g., brain, heart, liver lung, pancreas, colon) or a gland of a subject or a portion thereof (e.g., prostate, pituitary or mammary gland).

As used herein, the term "administering" to a subject includes dispensing, delivering or applying a composition of the invention to a subject by any suitable route for delivery of the composition to the desired location in the subject, including delivery by either the parenteral or oral route, intramuscular injection, subcutaneous/intradermal injection, intravenous injection, buccal administration, transdermal delivery and administration by the rectal, colonic, vaginal, intranasal or respiratory tract route.

A "biocompatible radionuclide" or "biocompatible radioisotope" is an isotope that is recognized as being useful for injection into a patient for nuclear medicine applications. Examples of biocompatible radionuclides include Iodine 123, Iodine 131, Gallium 67, Indium 111, Fluorine 18 and Technetium 99 m.

A "therapeutic radioisotope" is an isotope that is recognized as being useful for injection into a patient for treatment of disease. Examples of therapeutic radioisotopes include Palladium 103, Rhenium 186, Rhenium 188, Yttrium 90, Samarium 153, Gadolinium 159, or Holmium 166.

"Image" or "imaging" refers to a procedure that produces a picture of an area of the body, for example, organs, bones, tissues, or blood.

"Computed tomography (CT)" refers to a diagnostic imaging tool that computes multiple x-ray cross sections to produce a cross-sectional view of the vascular system, organs, bones, and tissues. "Positron emission tomography (PET)" and "single photon emission tomography" refers to a diagnostic imaging tool in which the patient receives a radioactive isotope by injection or ingestion which then, the radioactive signals from the isotope are detected by the PET camera and data is analyzed by computers to generate a 3D image. Different radionuclides are used to generate images with SPET and PET cameras. These radioactive isotopes are bound to compounds or drugs that are injected into the body and enable study of the physiology of normal and abnormal tissues. "Magnetic resonance imaging (MRI)" refers to a diagnostic imaging tool using magnetic fields and radiowaves to produce a cross-sectional view of the body including the vascular system, organs, bones, and tissues.

"Cancer" or "malignancy" are used as synonymous terms and refer to any of a number of diseases that are characterized by uncontrolled, abnormal proliferation of cells, the ability of affected cells to spread locally or through the bloodstream and lymphatic system to other parts of the body (i.e., metastasize) as well as any of a number of characteristic structural and/or molecular features. A "cancerous" or "malignant cell" is understood as a cell having specific structural properties, lacking differentiation and being capable of invasion and metastasis. Examples of cancers are, breast, lung, brain, bone, liver, kidney, colon, and prostate cancer, (see DeVita. et al., eds, Cancer Principles and Practice of Oncology, 6th. Ed., Lippincott Williams & Wilkins, Philadelphia, Pa., 2001; this reference is herein incorporated by reference in its entirety for all purposes).

The present invention therefore involves the production and use of oligonucleotide aptamers as binding ligands to PS found on apoptotic cells. The application of combinatorial chemistry coupled with polymerase chain reaction ("PCR") amplification techniques allow for the selection of small oligonucleotides from degenerate oligonucleotides libraries, which bind to target receptor molecules.

The use of aptamers as targeting agents offers several advantages compared with prior antibody techniques. The molecules are expected to penetrate the tissue much faster than whole antibodies, reach peak levels in the tissue sooner, and clear from the body faster thereby reducing toxicity to healthy tissues. In addition, the use of aptamers is expected to overcome the frequently encountered human anti-mouse antibody response. Also, the relatively small size of aptamers in comparison to whole antibodies favours their potential for greater tumour penetration, reduced immunogenicity, rapid uptake and faster clearance and makes them suitable vehicles for radionuclides and cytotoxic agents to be delivered to tumour. Aptamers that have very high affinity for targets are especially effective for a number of reasons. For example, a relatively small dose may be used in comparison with other treatments because the aptamers become localised in a target area, rather than being distributed throughout a body. Also, the effect of a labeling, therapeutic or diagnostic agent is enhanced by the high concentrations achieved within the target area.

In one embodiment, the present invention provides PS-specific aptamers which are either ribonucleic or deoxyribonucleic acid. In aspects the PS-specific aptamer is about 15 to about 90 nucleotides in length or any number therein between such as but not limited to 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 and 90 nucleotides. In a further embodiment, these ribonucleic or deoxyribonucleic acid aptamers are single stranded. In another embodiment, the present invention provides PS-specific aptamers comprising at least one chemical modification. In one embodiment, the modification is selected from the group consisting of: a chemical substitution at a sugar position; a chemical substitution at a phosphate position; and a chemical substitution at a base position, of the nucleic acid; incorporation of a modified nucleotide; 3' capping; conjugation to a high molecular weight, non-immunogenic compound; conjugation to a lipophilic compound; and phosphate backbone modification. In one embodiment, the non-immunogenic, high molecular weight compound conjugated to the PS-specific aptamer of the invention is polyalkylene glycol, preferably polyethylene glycol. In one embodiment, the backbone modification comprises incorporation of one or more phosphorothioates into the phosphate backbone. In another embodiment, the aptamer of the invention comprises the incorporation of fewer than 10, fewer than 6, or fewer than 3 phosphorothioates in the phosphate backbone. In one embodiment, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a PS-specific labeled aptamer comprising a nucleic acid sequence.

The PS-specific aptamers of the invention, or variations thereon, may be connected to another compound for various uses. An aptamer may be joined to a ligand, such as those disclosed herein, by, for example, ionic or covalent bonds, or by other ways such as hydrogen bonding. The aptamer may thus guide the ligand to the target. The aptamer is preferably directly connected to the ligand. More specifically, the aptamer may be bound to the ligand without the use of a peptide tether. An aptamer may be joined to a ligand or other agent by a pendant moiety such as an amino or hydroxyl group. Several other agents may be attached to the same aptamer, and several aptamers may be attached to the same agent.

In accordance with the present invention a suitable method for generating the PS-specific aptamer of the invention is with the process entitled "Systematic Evolution of Ligands by Exponential Enrichment" (SELEX™) generally depicted in Figure 1. The SELEX™ process is a method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules and is described in, e.g., U.S. Pat. No. 5,475,096 and U.S. Pat. No. 5,270,163 (see also WO 91/19813) entitled "Nucleic Acid Ligands"(the disclosures of which are incorporated herein by reference in their entirety). Each SELEX™-identified nucleic acid ligand, i.e., each aptamer, is a specific ligand of a given target compound or molecule. The SELEX™ process is based on the unique insight that nucleic acids have sufficient capacity for forming a variety of two- and three- dimensional structures and sufficient chemical versatility available within their monomers to act as ligands (i.e., form specific binding pairs) with virtually any chemical compound, whether monomeric or polymeric. Molecules of any size or composition can serve as targets. In the present invention the target molecule is phosphatidylserine.

SELEX™ relies as a starting point upon a large library or pool of single stranded oligonucleotides comprising randomized sequences. The oligonucleotides can be modified or unmodified DNA, RNA, or DNA/RNA hybrids. In some examples, the pool comprises 100% random or partially random oligonucleotides. In other examples, the pool comprises random ©r partially^andom oligonucleotides containing at least one fixed sequence and/or conserved sequence incorporated within randomized sequence. In other examples, the pool comprises random or partially random oligonucleotides containing at least one fixed sequence and/or conserved sequence at its 5^* and/or 3' end which may comprise a sequence shared by all the molecules of the oligonucleotide pool. Fixed sequences are sequences common to oligonucleotides in the pool which are incorporated for a preselected purpose such as, CpG motifs described further below, hybridization sites for PCR primers, promoter sequences for RNA polymerases (e.g., T3, T4, T7, and SP6), restriction sites, or homopolymeric sequences, such as poly A or poly T tracts, catalytic cores, sites for selective binding to affinity columns, and other sequences to facilitate cloning and/or sequencing of an oligonucleotide of interest. Conserved sequences are sequences, other than the previously described fixed - sequences, shared by a number of aptamers that bind to the same target.

The oligonucleotides of the pool preferably include a randomized sequence portion as well as fixed sequences necessary for efficient amplification. Typically the oligonucleotides of the starting pool contain fixed 5' and 3' terminal sequences which flank an internal region of 30-50 random nucleotides. The randomized nucleotides can be produced in a number of ways including chemical synthesis and size selection from randomly cleaved cellular nucleic acids. Sequence variation in test nucleic acids can also be introduced or increased by mutagenesis before or during the selection/amplification iterations.

The random sequence portion of the oligonucleotide can be of any length and can comprise ribonucleotides and/or deoxyribonucleotides and can include modified or non-natural nucleotides or nucleotide analogs. See, e.g., U.S. Pat. No. 5,958,691; U.S. Pat. No. 5,660,985; U.S. Pat. No. 5,958,691; U.S. Pat. No. 5,698,687; U.S. Pat. No. 5,817,635; U.S. Pat. No. 5,672,695, and PCT Publication WO 92/07065 (the disclosures of which is incorporated herein by reference). Random oligonucleotides can be synthesized from phosphodiester-linked nucleotides using solid phase oligonucleotide synthesis techniques well known in the art. See, e.g., Froehler et al., Nucl. Acid Res. 14:5399-5467 (1986) and Froehler et al., Tet. Lett. 27:5575-5578 (1986). Random oligonucleotides can also be synthesized using solution phase methods such as triester synthesis methods. See, e.g., Sood et al., Nucl. Acid Res. 4:2557 (1977) and Hirose et al., Tet. Lett., 28:2449 (1978). Typical syntheses carried out on automated DNA synthesis equipment yield 10¹⁴-10¹⁶ individual molecules, a number sufficient for most SELEX™ experiments. Sufficiently large regions of random sequence in the sequence design increases the likelihood that each synthesized molecule is likely to represent a unique sequence.

The starting library of oligonucleotides may be generated by automated chemical synthesis on a DNA synthesizer. To synthesize randomized sequences, mixtures of all four nucleotides are added at each nucleotide addition step during the synthesis process, allowing for random incorporation of nucleotides. As stated above, in one embodiment, random oligonucleotides comprise entirely random sequences; however, in other embodiments, random oligonucleotides can comprise stretches of nonrandom or partially random sequences. Partially random sequences can be created by adding the four nucleotides in different molar ratios at each addition step.

The starting library of oligonucleotides may be either RNA or DNA. In those instances where an RNA library is to be used as the starting library it is typically generated by transcribing a DNA library in vitro using T7 RNA polymerase or modified T7 RNA polymerases and purified. The RNA or DNA library is then mixed with the target under conditions favorable for binding and subjected to step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity. More specifically, starting with a mixture containing the starting pool of nucleic acids, the SELEX.™. method includes steps of: (a) contacting the mixture with the target molecules (PS liposomes) under conditions favorable for binding; (b) partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules; (c) dissociating the nucleic acid-target complexes; (d) amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids; and (e) reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific, high affinity nucleic acid ligands to the target molecule. In those instances where RNA aptamers are being selected, the SELEX™ method further comprises the steps of: (i) reverse transcribing the nucleic acids dissociated from the nucleic acid-target complexes before amplification in step (d); and (ii) transcribing the amplified nucleic acids from step (d) before restarting the process.

Within a nucleic acid mixture containing a large number of possible sequences and structures, there is a wide range of binding affinities for a given target. A nucleic acid mixture comprising, for example, a 20 nucleotide randomized segment can have 4²⁰ candidate possibilities. Those which have the higher affinity constants for the target are most likely to bind to the target. After partitioning, dissociation and amplification, a second nucleic acid mixture is generated, enriched for the higher binding affinity candidates. Additional rounds of selection progressively favor the best ligands until the resulting nucleic acid mixture is predominantly composed of only one or a few sequences. These can then be cloned, sequenced and individually tested for binding affinity as pure ligands or aptamers.

Cycles of selection and amplification are repeated until a desired goal is achieved. In the most general case, selection/amplification is continued until no significant improvement in binding strength is achieved on repetition of the cycle. The method is typically used to sample approximately 10¹⁴ different nucleic acid species but may be used to sample as many as about 10¹⁸ different nucleic acid species. Generally, nucleic acid aptamer molecules are selected in a 5 to 20 cycle procedure. In one embodiment, heterogeneity is introduced only in the initial selection stages and does not occur throughout the replicating process.

Thus in accordance with the present invention the PS-coated liposomes were used in conjunction with the SELEX™ procedure to generate PS-specific aptamers. The inventiorrincludes compositions, kits, and methods for assessing the presence of apoptotic cells in a sample using the PS-specific aptamers.

In an aspect of the present invention is a method for the in vivo imaging of cell death, e.g., cell death caused by apoptosis, in a mammalian subject, for example, in an organ of a mammalian subject or a portion thereof (e.g., brain, heart, liver, lung, pancreas, colon) or a gland of a mammalian subject or a portion thereof (e.g., prostate or mammary gland). The method includes administering to the subject an imaging composition comprising PS-specific aptamer coupled to a radioactive nuclide used for imaging purposes and obtaining an image, wherein said image is a representation of cell death in the mammalian subject. Non-invasive imaging of apoptosis can be done in vivo using such imaging modalities such as magnetic resonance imaging (MRI), single photon emission tomography (SPET) and positron emission tomography (PET).

The aptamer may be conjugated to a contrast agent such as , but not limited to, gadolinium and used to obtain a magnetic resonance image (MRI). In one embodiment, the magnetic resonance image is obtained 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120 minutes after the administration of the magnetic resonance imaging composition to the subject. In another embodiment, the magnetic resonance image is obtained about 12-30, 15-25, 20-25, or 20-30 hours after the administration of the magnetic resonance imaging composition to the subject. Ranges intermediate to the above recited values are also intended to be part of this invention. For example, ranges using a combination of any of the above recited values as upper and/or lower limits are intended to be included. In a preferred embodiment, the magnetic reasonance image is obtained at a plurality of time points, thereby monitoring changes in the number of cells undergoing cell death or monitoring changes in the location of cells undergoing cell death. In another embodiment, the magnetic reasonance imaging composition is administered intravenously, intraperitoneal^, intrathecal^, intrapleurally, intralymphatically, or intramuscularly.

The aptamer may be conjugated to imaging radionuclides for use in SPET which is a modality of nuclear imaging. Examples of SPET radionuclides include, but are not limited to, ^99mTc, ¹¹¹In, and ¹²³I. An aptamer conjugated to a SPET radionuclide may be administered n to a patient and the patient may then be imaged with a SPET camera to obtain an image of wherein the said image is a representation of cell death in the mammalian subject.

The aptamer may be conjugated to imaging radionuclides for use in PET which is another nuclear imaging modality. Examples of PET radionuclides include, but are not limited to, ¹⁸F, ¹¹C, ¹⁵O, and ¹²⁴I. An aptamer conjugated to a PET radionuclide may be administered to a patient and then the patient may be imaged with a PET camera to obtain an image of wherein the image is a representation of cell death in the mammalian subject.

In a further aspect, the present invention provides an optical imaging composition which comprises a PS-specific aptamer coupled to a biologically compatible and optically active molecule, such as a fluorescent dye like fluorescein, which can be visualized during optical evaluations such as endoscopy, brochoscopy, peritonoscopy, direct visualization, surgical microscopy and retinoscopy. In yet another aspect, the present invention provides a method for imaging cell death in a mammalian subject in vivo by administering to the subject an optical imaging composition comprising annexin coupled to an optically active molecule; illuminating the subject with a light source; and visually monitoring the presence of the optical imaging composition in the subject, thereby obtaining an image, wherein the image is a representation of cell death in the mammalian subject.

In another aspect, the present invention provides a composition comprising a PS- specific aptamer coupled with a therapeutic radioisotope, e.g., .¹⁰³ Pd, .¹⁸⁶ Re, ¹⁸⁸ Re, ^∞ Y, ¹⁵³Sm, ¹⁵⁹Gd, or ¹⁶⁶ Ho. The therapeutic radioisotope and the PS-specific aptamer may be coupled at a ratio of 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, or 2:1 (therapeutic radioisotope:PS-specific aptamer). Ranges of values using a combination of any of the above recited values as upper and/or lower limits are intended to be included in the present invention.

It is contemplated that that the labeled PS-specific aptamer compositions of the invention will be administered, in vivo. Therefore, it will be desirable to prepare the compositions of the invention as a pharmaceutical composition appropriate for the intended application. Generally this will entail preparing a pharmaceutical composition that is essentially free of pyrogens, as well as any other impurities that could be harmful to humans or animals. One also will generally desire to employ appropriate salts and buffers to render the complex stable and allow for complex uptake by target cells. Aqueous compositions of the present invention comprise an effective amount of the labeled PS-specific aptamer (or non-labeled PS-specific aptamer) dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions can also be referred to as inocula. The phrases "pharmaceutically or pharmacologically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or a human, as appropriate. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Supplementary contrast enhancing ingredients also can be incorporated into the compositions.

The term "effective amount" as used herein refers to any amount of the labeled PS-specific aptamer compositions of the invention that produce a reproducible and evaluable image of a given in vivo site. This may vary depending on the size of the subject, the site at which the composition is to be administered and the route of such administration.

Administration of the imaging compositions according to the present invention will be via any common route used in imaging so long as the target tissue is available via that route. This includes administration by orthotopic, intradermal subcutaneous, intramuscular, intraperitoneal, intrathecal, or intravenous injection. Alternatively, oral, nasal, buccal, rectal, vaginal or topical administration also are contemplated. For imaging atherosclerotic plaques intravenous injection is contemplated. Such injections compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients. For imaging of tumours, direct intratumoural injection, injection of a resected tumour bed, regional (i.e., lymphatic) or general administration is contemplated. It also may be desired to perform continuous perfusion over hours or days via a catheter to a disease site, e.g., a tumour or tumour site. The imaging compositions of the present invention are advantageously administered in the form of injectable compositions either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared.

The compositions of the invention can be sterilized by heat, radiation and/or filtration, and used as such, or the compositions can be further dehydrated for storage, for instance by lyophilization. For practical application the compositions of the invention in the medical field, it is contemplated that the dried components and the carrier liquid can be marketed separately in a kit form whereby the contrast agent is reconstituted by mixing together the kit components prior to injection into the circulation of patients. A typical composition for such purpose comprises a pharmaceutically acceptable carrier. For instance; pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like may be used. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyloleate. Aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, buffered solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial agents, antioxidants, chelating agents and inert gases. The pH and exact concentration of the various components the imaging composition may be adjusted according to well known parameters the amount and degree of signal intensity observed and required. The individual components of the labeled PS-specific aptamer imaging compositions of the present invention may be provided in a kit, which kit may further include instructions for formulating and/or using the imaging agents of the invention. The kit also may comprise a device for delivering the composition to a mammal. In particular embodiments, the compositions of the present invention may be used to assess the efficacy or dosing of a particular existing drug. For example, in the case of cancer, the tumour size or composition may be monitored prior to and after the administration of a given drug treatment to assess whether the treatment is effective at reducing the size or composition of the tumour.

The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific Examples. These Examples are described solely for purposes of illustration and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation^

Examples

Example One - SELEX Procedure (figure IK Trends in Anal. Chem. 2006. 25: 681-

691. Hamula et al.)

Step l

PS 10% Liposomes

90% PC, 10% PS. DPPC: DPPS is 1:0.1 molar ratio or 20:2 ratio. Use 20 mg/mL of DPPC and 1.03 mg/mL of DPPS. If use 1 mL of DPPC and 2 mL of DPPS at these concentrations you will have a 1:0.1 ratio. Add chloroform to the lipid solution if need to increase volume before rotovaping.

Typical Preparation:

1.2 mL DPPC (20 mg/mL)

2.4 mL DPPS (1.03 mg/mL)

1.2 mL Cholesterol (4.89 mg/mL)

^■ - Used plastic tips to measure out.

^■ Put into 250 mL round bottom flask.

^■ Put flask onto rotovap set at 210 rpm and 40⁰C, vacuum on. (Note: These parameters may be changed depending on the vacuum and rotovap used.) I have had evaporation completed between 15-30 minutes.

^■ When chloroform is evaporated, there should be a uniform lipid film on the flask. If the flask looks spotted, need to add chloroform and repeat rotovap step.

^■ Once a film is formed, add 2 mL sodium acetate IM, pH 6.0. Dissolve for approximately 30 minutes. Use a Pasteur pipette to wash sodium acetate over lipid film and redissolve lipids in sodium acetate.

^■ Put flask onto sonicator for 20 minutes. ■ Assemble extruder. Ensure it is clean. Use 2 um filters. Hot water hoses in water bath at 50⁰C. Pipette liposomes into extruder.

■ Extrude 1, change filter, extrude 2, change filter, extrude 3, 4, 5, 6, 7, 8, change filter and extrude 9.

■ Measure volume of liposomes. Can store in fridge at 4C.

■ Column separation. Use PD-IO desalting columns. Add 25 mL solvent (equilibration volume), HEPES 0.1 M, pH 7.5. Can fill column 5 times, which is approximately 25 mL Add liposome sample.

■ Add 6 mL HEPES buffer to elute.

■ Collect sample in 7 centrifuge tubes. Approximately 0.5 mL/tube.

^■ Either centrifuge liposome samples for 5 minutes at 10, 000 rpm, 20°C or 4°C or allow them to settle overnight stored in 4°C fridge.

^■ Use ZetaSizer in Pharmacy to determine the size of liposomes in each tube. If a tube does not have liposome pellet at bottom of tube, do not need to size that sample since it will not be used in experiments.

^■ Add 100 μL of liposomes to cuvet. Add 3 mL HEPES buffer. Use automatic setting. Keep size reports.

^■ Determine which liposome tubes/samples to use for experimental procedures. For SELEX typically need 1 mL liposomes for testing and ImL liposomes for control.

Step 2

Measure PS10% liposomes with spectrometer. Convert to number to cells. The turbidity of the solution tells us how dense the sample is. Beer's law Abs=abc. A=molar absorbtivity. B=light pathlength. C= concentration in M. Abs= -logT. Use this equation to calculate concentration of liposomes starting with. Start with ImL of liposomes. Sufficient form a pellet. Use 2mL cuvets in spectrometer. Use of A-600nm (absorbance). Note: 600nm was used to measure cells. Click set ref first, use buffer as a reference.

Step 3

Prepared selection buffer: (Blank et al). 5OmM Tris-HCI (ph 7.4), 5mM KCI, 10OmM NaCI (in lab), ImM MgCl2 , and 0.1% NaN3 . Use this except use 200mm NaCI. (Wang et al. Selection buffer: 5mM MgCI2 in PBS). To reduce background binding add bovine serum albumin (BSA). Selection buffer would include 0.2% BSA. (Could also add yeast tRNA). Note: Blank et aLused 5 times molar excess of BSA during selection rounds. If using 200pmol of aptamer then use lOOOpmol of BSA. Can also try increasing the amount of BSA in each selection round depending on how successful the selections are.

Combine DNA (Round 1 use library, other rounds use aptamer pool) and ImL of selection buffer (or less selection buffer, ie could be 400μl). Start with 2nmol DNA and later use 0.2nmol DNA. ssDNA pools were denatured by heating at 80⁰C for 10 minutes in selection buffer and renatured at 0°C (on ice) for 10 minutes before binding (Blank et al.). (Or by heating to 95°C for 5minutes and cooled to 0°C (on ice) in selection buffer [Wang et al]). Centrifuge liposome sample and remove buffer supernatant. Add DNA in selection buffer to liposome pellet. Vortex. Incubate the PS10% liposomes and DNA at 37°C for 45 minutes. Use two aliquots of liposomes. One with DNA and one with only selection buffer as control.

Step 4

Separate bound and unbound aptamers. Centrifuge liposome sample at 10, OOOrpm for 5 minutes at 20⁰C. Remove unbound DNA in supernatant. Wash liposomes three times with ImL high salt selection buffer. Wash for one minute. Keep washes. This will remove most of the unbound aptamers from liposomes.

To separate the bound aptamers from the liposomes. First going to try simply using a high to low salt gradient. Start with 20OmM NaCI selection buffer and then change to 2mM NaCI selection buffer. If this does not work, I will have to try another type of elution method (see paper by Wang et al.,). After washes, Resuspend liposomes and bound aptamers in a low salt concentration selection buffer. Use 2mM NaCI. Allow time to incubate. Centrifuge liposomes. The supernatant should contain pool of binding aptamers from this round of selection.

Step 5

PCR. Amplify the aptamers from the pool. Supernatant and control sample (no DNA). Could also amplify washes if want to.

PCR Program. Programmed into per in fourth floor lab. It is under #8. 94C for 5 minutes (note: could do for 2 minutes), 30sec at 94°C, 30sec at 57°C, 20sec at 72°C. Note: Omitted final extension of 72°C for 5 minutes. 18-22 cycles. End at 4°C. Can do up to 30 cycles. The cycling info below indicates this will work for KOD polymerase.

1 Final reaction concentrations of the components shown are for typical PCR reactions with the indicated DNA polymerases.

2 MgCI2 is included as a component of the 1OX PCR buffer for PfuTurbo and PfuULtra high-fidelity DNA polymerases.

3 Data not available. http://www.emdbiosciences.com/htmL/NVG/KOD Hot.htm

Standard reaction setup

2+ a To optimize for targets greater than 2kb, final Mg concentration may be adjusted to between 1.5 and 2.25 mM. b See Template DNA section.

Note: Use PCR grade water. Check concentrations of primers.

PCS cycling parameters

PCR Segment Number genomic *, or genomic, *, or λ or plssmtd DNA genomic DNA

•f cycles plasmid DMA plasmid DNA

De»a titration 1 S5*C 2 mm 9S*C, 2 mm 94*ζ 2 min 94'Q 2 min

Amplification 25-40 9S*C_»3©$ 94"Q iS s JMTC, 15 s

Primer Primer Primer Primer

0.,- 5J-Q 30 s (T.- 5K305 π,- (5-10)]^*C; 30s

7TC. 72^; I nAi 72-C, 20 $ per kbp 6B-C, 30 $ per kfep

1 minperkbp (targets* W⁾ ktø {targets < 21 kbp) or68*C 2 mm ptrkbp

final extension 1 72*C, 10 min 72^»ς t0 miB Omit Omit

Cycle number

The number of cycles (steps 2 through 4 in the above table) required to generate a PCR product will depend on the source and amount of starting template in the reaction, as well as the efficiency of the PCR as is understood by those of skill in the art. In general, 20-40 cycles will be adequate for a wide range of templates. It is common to use fewer cycles when amplifying targets from plasmids (i.e., subcloning) where a high number of copies of template is easily attained, as this reduces the chance of amplifying a mutation. A higher number of cycles (e.g., 40) may be necessary when amplifying from genomic DNA since the target sequence will be in low abundance.

Cycling conditions Temperature and time

The following table allows for primer extension that occurs during temperature ramping between steps.

_ „ 20-40 cycles. For more information see Cycle number Repeat steps 2-4 , , below

Step 6

PAGE gel. Vertical native PAGE gels. Biorad protean III gel. Run a gel of amplified aptamers, DNA library and ladder.

Recipe for 1OmLs of 7.5% page gel:

^■ 7 mL 1 x TAE

■ 3mL 40% Acrylamide/Bis (19:1)

■ 65uL 20% or 10% APS

^■ 13 uL TEMED

Procedure:

DNA samples were kept on ice (library, ladder and aptamers). Set up 2 casting gels. Clamp in. Put parafilm on top of foam. Combine chemicals for gel. Add gel using pipet and pour it in to top. Put gel into case. Need two in there. Fill middle chamber with TAE xl. Use lug of DNA ladder and 0.3 ug DNA sample. Last time used 2μL of DNA and 0.5μL buffer and 4uL DNA and lμL buffer for both the library and the ladder. Only the 4μL DNA samples were visible on the gel. May want to add more buffer so easier to see. Mix DNA and loading buffer together on parafilm with pipet. Add samples to gel. Use 80 volts. Can use 100-110 volts to go faster. Run for 1-2 hours.

When gel is done remove it from system. Prepare 1% ethydium bromide stock in water. Add lOuL of the stock solution to 10OmL total volume water/buffer for gel staining. Put gel in Tupperware. Incubate. Put gel onto imager. Take picture.

Step 7

After PCR amplification need to purify aptamers. Take aptamer sample. Use Qiagen minilute PCR purification kit. This removes the primers.

Step 8 Purify.

1) Heat to 94°C. To separate 2 DNA strands.

Example 2 - AnnexinV-Liposome Protocol

1) Centrifuge PC, 5%PS and 10%PS lipsomes(chosen samples by size). 5 minutes at 10,000 rpm, 20⁰C. Remove supernatant and add 0.5ml binding buffer to liposomes. Vortex. Centrifuge again, remove supernatant and add 50OuI binding buffer.

2) Transfer lOOμl of PC liposomes to 3 centrifuge tubes.

3) Transfer lOOμl of 5% PS liposomes to 3 centrifuge tubes. 4) Transfer lOOμl of 10% PS liposomes to 3 centrifuge tubes.

5) Add 3ul Annexin V to 2 of 3 liposomes tubes. Do for each sample of liposomes.

6) Put all tubes into drawer and incubate in dark for 15 minutes.

7) Add 40OuI binding buffer (or hepes buffer) to each of the tubes. The binding buffer control tubes will add hepes buffer instead of binding buffer.

8) Try this step. Centrifuge all of the tubes. If does not separate, go to step 9. If does separate, remove supernatant and add 500μl of either binding buffer or hepes buffer.

9) Transfer 60μl of liposome sample into falcon tube. Add lOOOμl of either binding buffer or hepes buffer to falcon tubes to dilute sample for FACS.

10) Take samples to FACS.

Example 3 - Representative SELEX Procedure/Results, target phosphatidylserine (PS)- liposomes

Round 0 preparing first round

primers and template testing

PCR amplification

Volume: 1000 μL

# of cycles: 3 eps file: cde.eps eps file 2: rOpcr.eps denaturing using 250 mM NaOH eluted top strand in 300 mM NaCI, 30 mM EDTA precipitated in EtOH, resuspend in 1 mL dH2O

A(260) 0.017

Dilution 100 concentration 2.12288E-06 moles 2.12288E-09 molecules 1.2784E+15

Round 1

Incubation of ssDNA pool with liposomes using 2x13 mL centrifuge tubes Conditions per tube:

2.4 mL 5xSELEX buffer

8.0 mL dH2O 0.5 mL ssDNA pool RUN

0.1 mL 100 mM spermidine

heat in waterbath (set to 90⁰C) for 5 min slowly cool down (30min) in 37⁰C walkin incubator heat up beads to 37 ⁰C in waterbath (20min)

add 1.0 mL beads to 13 mL tube incubation: 3.0 hours precipiate liposomes by centrifugation

5 krpm for 10 min

1st PCR of liposomes without further purification labbookpages: 39-41 eps file: rlpcr.eps agarose gel: p.41

Round 1 (continued)

RlOUT: Phenol-Chlorophorm, Ethanol ppt.

PCR: 3.0 mL cycles: 16 equals 65,536 eps file: pcrrlrl.eps eps file 2: rloutrlg.eps precipitated 3 mL of PCR in 300 mM NaCI, 10 mM EDTA spin down and resuspend in 500 μL TE #2 buffer add 100 μL PCR p47 (hot PCR) add 100 μL 1 M NaOH

45⁰C 15 min add 2xformamide loading dye

99⁰C 5 min load onto 10 % gel, 20 W, 2.5 hours resuspended in 200 μL TE#2 (-80⁰C freezer box R2IN) Round 2

Incubation of ssDNA pool with PS-liposomes using 2x1.5 mL centrifuge tubes

Conditions per tube:

240 μL 5xSELEX buffer p.21

800 μL dH2O

50 μL ssDNA pool R2IN

10 μL 100 mM spermidine

heat in waterbath (set to 90⁰C) for 5 min

TURN OFF WATERBATH AND PLCAE TUBES IN RACK INSIDE slowly cool down (30min) in 37⁰C incubator heat up beads to 37 ⁰C in waterbath (20min)

100 μL beads add to 1.5 mL tube incubation: 3.0 hours

Centrifuge: 12 krpm for 5 min Phenol - Chlorophorm - Ethanol ppt.

PCR large scale: 1 mL

Cycles: 12

Round 3

Incubation of ssDNA pool with liposomes using 2x1.5 mL centrifuge tubes

Conditions per tube:

250 μL 5xSELEX buffer p.21

685 μL dH20

5 μL ssDNA pool R3IN

10 μL 100 mM spermidine

heat in waterbath (set to 90⁰C) for 5 min TURN OFF WATERBATH AND PLCAE TUBES IN RACK INSIDE slowly cool down (30min) in 37⁰C walkin incubator heat up beads to 37 ⁰C in waterbath (20min)

50 μL control liposomes add to 1.5 mL tube incubation: 15 min

Centrifuge: 12 krpm for 2 min collect supernatante

heat in waterbath (set to 90⁰C) for 5 min

50 μL liposomes (pos) add to 1.5 mL tube incubation: 40 min

Centrifuge: 12 krpm for 2 min

Phenol - Chlorophorm - Ethanol ppt.

PCR large scale:

Cycles: 15

Round 3R

Incubation of ssDNA pool with liposomes using 1.5 mL screw-cap centrifuge tubes

Conditions per tube:

200 μL 5xSELEX buffer p.21

725 μL dH20

5 μL ssDNA pool R3IN

10 μL 100 mM spermidine

10 μL IM glycine

heat in waterbath (set to 90⁰C) for 5 min

TURN OFF WATERBATH AND PLCAE TUBES IN RACK INSIDE slowly cool down (30min) in 37⁰C walkin incubator heat up beads to 37 ⁰C in waterbath (20min)

50 μL 1% PS liposomes incubation: 15 min

Centrifuge: 12 krpm for 2 min collect supernatante

Phenol-Chlorophorm-Ethanol ppt. resuspend in 100 μL #2 TE buffer

PCR 5 μL for PCR cycles needed: 15

Round

4R

Incubation of ssDNA pool with liposomes using 1.5 mL screw-cap centrifuge tubes

Conditions per tube:

200 μL 5xSELEX buffer p.21

720 μL dH20

10 μL ssDNA pool R4IN

10 μL 100 mM spermidine

10 μL IM glycine

heat in waterbath (set to 90⁰C) for 5 min

50 μL 1% PS liposomes incubation: 15 min

Centrifuge: 12 krpm for 2 min collect supernatante

resuspend pellet in 1 mL of Wash buffer p.84 (WB) WB = IxSELEX buffer p.21 + 40 mM KCL + 10 mM GIy pellet 12 krpm for 2 min Phenol-Chlorophorm-Ethanol ppt. resuspend in 50 μL #2 TE buffer PCR 5 μL for PCR cycles needed: 20

lane 2/3 10 μL Supernatante lane 4/5 10 μL S Washl (WB) lane 6/7 5 μL P-C-E R4Rout

Round

5R

Incubation of ssDNA pool with liposomes using 1.5 ml. screw-cap centrifuge tubes

Conditions per tube:

200 μL 5xSELEX buffer p.21

720 μL dH20

10 μL ssDNA pool R5IN

10 μL 100 mM spermidine

10 μL IM glycine

heat in waterbath (set to 90⁰C) for 5 min

50 μL 1% PS liposomes incubation: 15 min

Centrifuge: 12 krpm for 2 min collect supernatante

resuspend pellet in 1 mL of Wash buffer p.84 (WB) WB = IxSELEX buffer p.21 + 40 mM KCL + 10 mM GIy pellet 12 krpm for 2 min Phenol-Chlorophorm-Ethanol ppt. resuspend in 50 μL #2 TE buffer

PCR 5 μL for PCR cycles needed:

lane 2/3 5 μL Supernatante lane 4/5 5 μL S Washl (WB) lane 6/7 5 μL P-C-E R5Rout lane 8/9 5 μL dH2O lanes 8/9 empty

Example 4 - Selection of Aptamers binding to apoptotic cells done using the SELEX procedure.

The target used was phosphatidylserine (PS). Liposome constructs that contain various amounts of PS were successfully prepared and analyzed and demonstrated to be used to generate PS-specific aptamer. The liposome constructs can be manipulated in ways similar to apoptotic cells and used to perform a modified cell-SELEX type procedure. The advantage of using a liposome construct containing PS is that the liposomes are stable for up to 8 days whereas apoptotic cells are not stable due to the biochemical process of apoptosis breaking down the cell in a systematic manner. The SELEX library was a DNA library with a 43 nucleotide random region. The randomized region of the library allowed to have a large variety of possible sequences that can bind to the target. The SELEX procedure allowed the isolation of sequences that bind with high affinity to the target, PS.

The DNA gel images are all agarose gels with DNA stained by ethidium bromide.

Results:

Round 0. This round was to prepare for future rounds of selection. Test the primers and template for proper PCR amplification. PCR amplified the pool in three cycles. Denatured the pool using 250 mM NaOH. Separated the top strand and the bottom strand to prepare the ssDNA pool. Figure 2. indicates PCR amplification of DNA library. Lane 1: 100 bp marker, lane 2: single stranded DNA library, lanes 3-5: cycles 1-3.

Round 1. Incubate the ssDNA pool from Round 0 with PS 10% liposomes for 3 hours at 37 °C using our SELEX buffer. Centrifuge the liposomes and DNA. The liposomes and bound DNA will pellet. Phenol-chloroform-ethanol purify the DNA from the liposome pool. PCR amplify the DNA pool. Figure 3 shows ael image of PCR amplified DNA pool from Round 1. Lane on far left is DNA ladder. Lane 2 is 10 cycles, lane 3 is 13 cycles and lane 4 is 15 cycles of PCR amplification.

Round 2. Incubated the ssDNA pool from Round 1 with PS 10% liposomes for 3 hours at 37 "C using our SELEX buffer. Centrifuge the liposomes and DNA. The liposomes and bound DNA will pellet. Phenol-chloroform-ethanol purify the DNA from the liposome pool. PCR amplify the DNA pool. Figure 4 shows a gel image of PCR amplified DNA pool from Round 2. Lane 1 (far left) is DNA ladder. Lane 2 is the material out of Round 2 after 12 cycles of PCR amplification.

Round 3. Incubate the ssDNA pool from Round 2 with PS 10% liposomes for 15 minutes at 37 ⁰C using our SELEX buffer. Centrifuge the liposomes and DNA. The liposomes and bound DNA will pellet. Phenol-chloroform-ethanol purify the DNA from the liposome pool. PCR amplify the DNA pool. Figure 5 shows a gel image of PCR amplified DNA pool from Round 3. Two lanes on left are DNA ladder. The numbers 15, 18, 20, and 22 indicate the number of cycles in the PCR reaction.

Round 3R. Incubated the ssDNA pool from Round 3 with PS 1% liposomes for 15 minute at 37 ^βC using our SELEX buffer and added glycine to improve binding. Centrifuge the liposomes and DNA. The liposomes and bound DNA will pellet. Phenol-chloroform- ethanol purify the DNA from the liposome pool. PCR amplify the DNA pool. Figure 6 shows an agarose gel of PCR amplified DNA pool from Round 3R, where DNA is stained with ethidium bromide. Lane 1: 15 cycles of PCR, lane 2: 18 cycles, lane 3: 20 cycles and lane 4: 22 cycles (far right lane).

Round 4R. Incubated the ssDNA pool from Round 3R with 1% PS liposomes for 15 minutes at 37 "C using our SELEX buffer and added glycine to improve binding. Centrifuge the liposomes and DNA. Wash the liposome pellet once. The liposomes and bound DNA will pellet. Phenol-chloroform-ethanol purify the DNA from the liposome pool. PCR amplify the DNA pool. Figure 7 shows PCR amplified DNA pool from Round 4R. The left lane is the DNA ladder. The numbers 15 and 19 indicate the number of cycles in the PCR reaction. Lane 2/3 are supernatant. Lane 4/5 are wash 1. Lanes 6/7 are the DNA bound to the pellet.

Round 5R. Incubated the ssDNA pool from Round 4R with 1% PS liposomes for 15 minutes at 37 C using our SELEX buffer and added glycine to improve binding. Centrifuge the liposomes and DNA. Wash the liposome pellet once. The liposomes and bound DNA will pellet. Phenol-chloroform-ethanol purify the DNA from the liposome pool. PCR amplify the DNA pool. Figure 8 shows PCR amplified DNA pool from Round 5R. Lane 1 (on left) is DNA ladder. Lane 2/3 is supernatant with 15 and 20 cycles, respectively. Lane 4/5 is wash 1 with 15 and 20 cycles, respectively. Lane 6/7 are DBA bound to the pellet 15 and 20 cycles of PCR amplification, respectively.

Round 6R. Incubated the ssDNA pool from Round 5R with 1% PS liposomes for 15 minutes at 37 "C using our SELEX buffer and added glycine to improve binding. Centrifuge the liposomes and DNA. Wash the liposome pellet once. The liposomes and bound DNA will pellet. Phenol-chloroform-ethanol purify the DNA from the liposome pool. PCR amplify the DNA pool. Figure 9 shows PCR amplified DNA pool from Round 6R. Lane 1 (on left) is DNA ladder. Lane 2/3 is PCR amplified water. Lane 4/5 is supernatant. Lane 6/7 are wash 1. Lane 9/10 are DNA bound to the pellet. The numbers 15 and 20 indicate the number of cycles in the PCR reaction.

Round 7R. Incubated the ssDNA pool from Round 6R with 1% PS liposomes for 15 minutes at 37 ^*C using our SELEX buffer and added glycine to improve binding. Centrifuge the liposomes and DNA. Wash the liposome pellet three times. The liposomes and bound DNA will pellet. Phenol-chloroform-ethanol purify the DNA from the liposome pool. PCR amplify the DNA pool. Figure 10 shows PCR amplified DNA pool from Round 7R. Lane 1 is supernatant after 15 cycles of PCR. Lane 2 is supernatant after 20 cycles of PCR. Lane 3 is Wash 1 after 15 cycles of PCR. Lane 4 is Wash 1 after 20 cycles of PCR. Lane 5 is Wash 2 after 15 cycles of PCR. Lane 6 is Wash 2 after 20 cycles of PCR. Lane 7 is Wash 3 after 15 cycles of PCR. Lane 8 is Wash 3 after 20 cycles of PCR. Lane 9 is pellet DNA after 15 cycles of PCR. Lane 10 is pellet DNA after 20 cycles of PCR.

Round 8R. Incubated the ssDNA pool from Round 7R with 1% PS liposomes for 15 minutes at 37 °C using our SELEX buffer and added glycine to improve binding. Centrifuge the liposomes and DNA. Wash the liposome pellet twice. The liposomes and bound DNA will pellet. Phenol-chloroform-ethanol purify the DNA from the liposome pool. PCR amplify the DNA pool. Figure 11 shows PCR amplified DNA pool from Round 8R. Lane 1 is DNA ladder. Lane 2/3 is supernatant. Lane 4/5 is wash 1. Lane 6/7 is Wash 2. Lane 8/9 are DNA bound to the pellet, with the left lane of each pair representing 15 cycles of PCR amplification, while the right lane of each pair represents 20 cycles of PCR.

Round 9R. Incubated the ssDNA pool from Round 8R with 1% PS liposomes for 15 minutes at 37 ^βC using our SELEX buffer and added glycine to improve binding. Centrifuge the liposomes and DNA. Wash the liposome pellet twice. The liposomes and bound DNA will pellet. Phenol-chloroform-ethanol purify the DNA from the liposome pool. PCR amplify the DNA pool. Figure 12 shows PCR amplified DNA from Round 9R. Lane 1 is RO supernatant. Lane 2 is RO wash 1. Lane 3 is RO bound to pellet. Lane 4 is empty. Lane 5 is R9 supernatant. Lane 6 is R9 wash 1. Lane 7 is R9 bound to pellet. Compared intensity of binding of RO and R9 aptamer pool and more binding in pellet to PS liposomes in DNA pool from R9. Figure 13 is a graph comparing binding of pool RO and R9R to liposomes containing PS. The graph demonstrates that the aptamers produced by the method of the present invention bind specifically to PS.

In conclusion, the SELEX experiments were completed and have selected a pool of aptamers that bind to phosphatidylserine. The aptamers of the invention are then used to determine dissociation constant values. Aptamers with the best binding to the PS target (lowest dissociation constant value) are used for further investigation in cell models of apoptosis. The selected aptamers are then radiolabled for use as an imaging agent.

Claims

Claims:

1. An aptamer that is specific and binds to phosphatidγlserine (PS).

2. The aptamer of claim 1, wherein said aptamer is RNA or DNA.

3. The aptamer of claim 1 or 2, wherein said aptamer is up to about 90 nucleotides.

4. The aptamer of claim 3, wherein said aptamer is about 15 to 90 nucleotides.

5. The aptamer of claim 3, wherein said aptamer is about 10 to 15 kDa.

6. The aptamer of claim 1, wherein said aptamer further comprises an imaging probe.

7. The aptamer of claim 6, wherein said probe is a biocompatible radionuclides selected from Iodine 123, Iodine 131, Gallium 67, Indium 111, Fluorine 18 and Technetium 99 m.

8. The aptamer of claim 6, wherein said probe is a therapeutic radioisotope for injection into a patient selected from radioisotopes include Palladium 103, Rhenium 186, Rhenium 188, Yttrium 90, Samarium 153, Gadolinium 159 and Holmium 166.

9. The aptamer of claim 3, wherein said aptamer comprises at least one chemical modification selected from the group consisting of: a chemical substitution at a sugar position; a chemical substitution at a phosphate position; a chemical substitution at a base position, of the nucleic acid; incorporation of a modified nucleotide; 3¹ capping; conjugation to a high molecular weight, non-immunogenic compound; conjugation to a lipophilic compound; and a phosphate backbone modification.

10. The aptamer of claim 9, wherein said modification is conjugation to polyalkylene glycol.

11. The aptamer of claim 9, wherein said backbone modification comprises incorporation of one or more phosphorothioates into the phosphate backbone.

12. The aptamer of claim 9, wherein said modification is the incorporation of fewer than 10, fewer than 6, or fewer than 3 phosphorothioates in the phosphate backbone.

13. The aptamer of claim 3, wherein said aptamer is joined to a ligand by ionic or covalent bonds, or by hydrogen bonding.

14. The aptamer of claim 13, wherein said aptamer is joined by a peptide tether.

15. The aptamer of claim 3, wherein said aptamer is joined to an agent by a pendant moiety selected from an amino or hydroxyl group.

16. The aptamer of any one of claims 1 to 15, wherein said aptamer is bound to a phosphatidylserine (PS)-liposome.

17. The aptamer of claim 16, wherein said liposome comprises up to about 40% phosphatidylserine.

18. The aptamer of any one of claims 1 to 17, provided as an aqueous composition.

19. A method of imaging a disease or condition involving apoptosis in a subject, the method comprising the step of administering the aptamer of claim 18 to said subject and imaging said subject.

20. A method for targeting or imaging a tissue in a subject comprising administering to the subject an aptamer of claim 6, and imaging said subject at various time points after said administration.

21. A method of determining the effectiveness of a cancer treatment in a subject, said method comprising the step of administering the aptamer of claim 18 to said subject undergoing a cancer treatment and imaging said subject at various time points after said cancer treatment.

22. A screening method for the identification of agents which modulate, either directly or indirectly, apoptosis of a cell, the method comprising, i) providing at least one candidate agent to be tested to a population of cells; ii) providing a phosphatidylserine specific labeled aptamer to said population of cells, and iii) determining the effect, or not, of said agent on apoptosis of said cells.

23. An imaging composition comprising a PS-specific aptamer having an imaging probe attached thereto, and an aqueous vehicle.

24. A method for generating DNA or RNA aptamers specific to phosphatidylserine (PS), the method comprising:

(a) contacting a mixture of randomly generated nucleic acid sequences of fixed length flanked by constant V and 3^ ends with target molecules (PS liposomes) under conditions favorable for binding;

(c) dissociating the nucleic acid-target complexes;

25. The method of claim 24, wherein RNA aptamers are being generated and further comprising the steps of: (i) reverse transcribing the nucleic acids dissociated from the nucleic acid-target complexes before amplification in step (d); and

(ii) transcribing the amplified nucleic acids from step (d) before restarting the method.

26. An aptamer produced by the method of claim 24 or 25.