TW202600828A - Drug-conjugated aptamer therapeutics and applications thereof - Google Patents

Abstract

The present disclosure relates generally to the field of nucleic acids and more particularly to aptamer(s) conjugating with an therapeutic agent to form an aptamer therapeutics.

Description

Appropriate therapies for drug combination and their applications

This disclosure pertains broadly to the field of nucleic acids, and more specifically to aptamers that combine with therapeutics to form aptamer therapies.

The blood-brain barrier (BBB) dynamically regulates brain homeostasis. It consists of capillary endothelial cells, outer cells, astrocyte foot processes, and nerve endings terminating on the capillary surface. Tight and adhesive junctions between adjacent endothelial cells prevent the paracellular transport of hydrophilic compounds across the BBB, and only lipophilic or water-soluble molecules smaller than 500 Da can be transported across cells via passive diffusion. Furthermore, active efflux transporters on the BBB limit the entry of chemotherapeutic agents into the central nervous system (CNS). Therefore, there is a need to develop chemotherapeutic agents capable of penetrating the BBB.

This disclosure discloses the development of aptamers that can be combined with anticancer drugs to form aptamer therapies for the treatment of cancer. In particular, these aptamer therapies can penetrate the BBB to treat brain cancer.

In one embodiment, this disclosure provides an isolated aptamer comprising a polynucleotide sequence selected from the group consisting of: ACGCTCGGATGCCACTACAGGTCGGCAATGCATGGTAACTAGTGCGGGTGTGTGCAACTCCTCATGGACGTGCTGGTGAC (SEQ ID NO:1); ACGCTCGGATGCCACTACAGGGGGTAGGTTCGCCGGGTCCAAATGCCTATTATCATCCAACTCATGGACGTGCTGGTGAC (SEQ ID NO:2); ACGCTCGGATGCCACTACAGCTCCATACGTATCTCCACCATTCCTTGGGGATTTATAAGTCTCATGGACGTGCTGGTGAC (SEQ ID NO:3); ACGCTCGGATGCCACTACAGATGTTCCAACTATAGTTGGGGTCAAATCTTCCCAATGGTGCTCATGGACGTGCTGGTGAC (SEQ ID NO:4); GTCGGCAATGCATGGTAACTAGTGCGGGTGTGTGCAACTC (SEQ ID NO:5); GGGGTAGGTTCGCCGGGTCCAAATGCCTATTATCATCCAA (SEQ ID NO:6); CTCCATACGTATCTCCACCATTCCTTGGGGATTTATAAGT (SEQ ID NO:7); and ATGTTCCAACTATAGTTGGGGTCAAATCTTCCCAATGGTG (SEQ ID NO:8); or a variant thereof or a salt of any of the foregoing.

In one embodiment, the polynucleotide sequence of SEQ ID NO:1 to 8 or a variant thereof or a salt thereof has a G-tetramer structure.

In one embodiment, the aptamer comprises a polynucleotide sequence of SEQ ID NO:1, 3, 5, or 7, or a variant thereof, or a salt of any of the foregoing. Preferably, the aptamer comprises a polynucleotide sequence of SEQ ID NO:1, or a variant thereof, or a salt of any of the foregoing.

In one embodiment, the polynucleotide sequence of SEQ ID NO:1 or 5 or a variant thereof or a salt of any of the foregoing forms 3 stem loops having a total of 9 GC pairs.

In one embodiment, this disclosure provides a method for delivering a drug to penetrate an individual's BBB, comprising administering to the individual an aptamer-therapeutic conjugate as described herein, wherein the aptamer-therapeutic conjugate is a combination of one or more of the therapeutics described herein and one or more aptamers described herein. Preferably, the aptamer has a G-tetramer structure and comprises a polynucleotide sequence having fewer than 150 base pairs.

In another embodiment, the aptamer comprises a polynucleotide sequence having fewer than 100 base pairs or about 50 to 100 base pairs.

In some embodiments, the aptamer used in the method comprises a polynucleotide having the sequence SEQ ID NO:1, 2, 3, 4, 5, 6, 7 or 8 or a variant thereof or a salt thereof.

In another embodiment, the aptamer used in the method comprises a polynucleotide having the sequence of SEQ ID NO:1, 3, 5, or 7, or a variant thereof, or a salt of any of the foregoing. Preferably, the aptamer comprises a polynucleotide sequence of SEQ ID NO:1 or 3, or a variant thereof, or a salt of any of the foregoing.

In some embodiments, variants of the aptamer described herein comprise at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of a unique region of any sequence selected from the group of SEQ ID NO: 1 to 8, or a variant thereof, or a salt thereof. In another embodiment, variants of the aptamer comprise at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of a nucleotide sequence selected from the group of SEQ ID NO: 1 to 8, or a variant thereof, or a salt thereof. In one particular embodiment, a variant of the aptamer comprises a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a unique region of any sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, and 7, or a variant thereof, or a salt thereof. In another embodiment, a variant of the aptamer comprises a nucleotide sequence of 80 consecutive nucleotides identical to any sequence selected from the group consisting of SEQ ID NO: 1 to 8, or a variant thereof, or a salt thereof, comprising 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 40, 30, 20, or 10 consecutive nucleotides. In another embodiment, a variant of the aptamer comprises a nucleotide sequence of 80 consecutive nucleotides consistent with the sequence of SEQ ID NO:1 or 5 provided, or a variant thereof, or a salt of any of the foregoing, comprising 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 40, 30, 20, or 10 consecutive nucleotides.

In some embodiments, variants of the aptamers described herein include chemical modifications selected from the group consisting of: chemical substitution at the sugar position of the nucleic acid sequence; chemical substitution at the phosphate position; and chemical substitution at the base position. In some embodiments, the modification is selected from the group consisting of: incorporation of a modified nucleotide; 3' capping; binding to a high molecular weight non-immunogenic compound (such as polyethylene glycol (PEG)); binding to a lipophilic compound; and modification of the phosphate backbone.

In one embodiment, this disclosure provides an aptamer-therapeutic agent complex comprising one or more therapeutic agents bound to one or more aptamers described herein.

In one embodiment, this disclosure provides a pharmaceutical composition comprising an aptamer-therapeutic combination and a pharmaceutically acceptable carrier or excipient.

In one embodiment, the treatment is a medication related to a CNS disease. In some embodiments, the CNS disease is a CNS tumor or neurodegenerative condition, such as neurodegenerative diseases and diseases related to CNS damage (such as stroke).

In some implementations, the treatment is an anticancer agent or a drug used for neurodegenerative diseases or CNS damage.

In one embodiment, the anticancer agent is a chemotherapy drug. In some embodiments, the chemotherapy drug is an alkylating agent, an antimetabolite, an anthraquinone, an antitumor antibiotic, a topoisomerase inhibitor, or a mitotic inhibitor.

Certain embodiments of anticancer agents include, but are not limited to, platinum-based chemotherapy drugs (such as cisplatin, oxaliplatin, and carboplatin), busulfan, carmustine, melphalan, lomustine, 5-fluorouracil, methotrexate, daunorubicin, doxorubicin, idarubicin, epirubicin, mitoxantrone, mitomycin, cabazitaxel, docetaxel, or paclitaxel. In another embodiment, the anticancer drug is a platinum-based drug.

In one embodiment, this disclosure provides a method for treating or preventing diseases in the CNS, comprising administering to an individual the adaptor-therapeutic agent combination described herein, wherein the therapy is a drug for treating CNS diseases.

In one embodiment, this disclosure provides a method for treating cancer comprising administering to an individual an aptamer-therapeutic agent complex as described herein, wherein the therapeutic agent is an anticancer agent.

In one embodiment, this disclosure provides a method for treating or preventing neuropathies, comprising administering to an individual the adaptor-therapeutic agent combination described herein, wherein the therapeutic agent is a drug for neuropathies.

In some embodiments, neurodegenerative diseases include neurodegenerative diseases, neuromuscular diseases (such as muscular atrophy, amyotrophic lateral sclerosis), brain diseases (such as attention deficit hyperactivity disorder (ADHD), Bell's palsy, carpal tunnel syndrome, cerebral aneurysm, diabetic neuropathy, epilepsy, migraine and headache syndrome, stroke and traumatic brain injury), and spinal diseases (such as spina bifida, spinal cord injury and spinal muscular atrophy).

In some implementations, neurodegenerative diseases include Alzheimer's disease, ataxia, Huntington's disease, Parkinson's disease, motor neuron disease, Lewy body disease, spinal muscular atrophy, multiple systemic atrophy and progressive supranuclear palsy, mild cognitive impairment, migraine, multiple sclerosis, myasthenia gravis, or amyotrophic lateral sclerosis (ALS).

Examples of aptamer-therapeutic combination compounds are those described herein.

In some implementations, the disease is CNS cancer, neurodegenerative disease, or a disease related to CNS damage (such as stroke).

In another embodiment, CNS cancer is brain cancer, brain metastasis, degenerative astrocytoma, neuroglioblastoma, or pia mater carcinoma.

In another embodiment, the pia mater carcinoma is lung cancer pia mater carcinoma (LM).

In another embodiment, the method used to treat and/or prevent neuropathies achieves systemic therapeutic effects, such as anticancer effects. Preferably, the method reduces the dosage of the drug. In some embodiments, the drug is an anticancer drug, such as a platinum-based drug.

Priority Information This application claims the interest and priority of U.S. Provisional Patent Application No. 63/626,192, filed January 29, 2024, the contents of which are incorporated herein by reference in their entirety. Sequence List

This application contains a sequence list, which has been submitted electronically in XML format and is hereby incorporated by full reference. The XML copy was created on January 28, 2025, named G4590-19000PCT_20250128_SeqListing.xml, and is 14,971 bytes in size.

Any methods, compounds, and materials similar to or equivalent to those described herein may be used in the practice of this invention. Unless otherwise defined, all technical terms, symbols, and other scientific terms or proper nouns used herein are intended to have the meanings commonly understood by those skilled in the art to which this invention pertains. In some cases, for clarity and/or convenience of reference, terms are defined herein with their commonly understood meanings, and the inclusion of such definitions herein should not necessarily be construed as indicating a substantial difference from the content generally understood in this art. Many techniques and procedures described or mentioned herein are well understood and commonly used by those skilled in the art using known methods.

Unless the context clearly requires otherwise, the singular forms “a”, “an”, and “the” include plural references. For example, the term “cell” includes one or more cells, including mixtures thereof. “A and/or B” is used herein to include all of the following alternatives: “A”, “B”, “A or B”, and “A and B”.

The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides, their polymers, or their complementary sequences, in single-stranded, double-stranded, or multi-stranded form. The term "polynucleotide" refers to a linear arrangement of nucleotides. Furthermore, "nucleotide" usually refers to a single unit of a polynucleotide, essentially a monomer. Nucleotides can be ribonucleotides, deoxyribonucleotides, or modified variants thereof.

As used herein, the term "aptamer" refers to a polynucleotide (such as a short nucleotide or deoxyribonucleotide) that binds to proteins, peptides, and small molecules with high affinity and specificity. An aptamer can also be described as a target-binding entity based on a polynucleotide. It can be composed of RNA or DNA and can employ secondary or tertiary structures, allowing it to fold into various complex molecular configurations.

The terms "compound," "biocompound," and "biocompound linker" refer to a connection between atoms or molecules. This connection can be direct or indirect. For example, a compound formed between a first part and a second part can occur directly through covalent bonds or linkers, or indirectly through non-covalent interactions. Such non-covalent interactions can include electrostatic forces (such as ionic bonds, hydrogen bonds, or halogen bonds), van der Waals forces, hydrophobic interactions, and other similar interactions.

The term "percentage of identity" refers to sequences or subsequences that are identical or have a specified percentage of identical amino acid residues or nucleotides. This percentage can range from approximately 80% to 99% or higher, with preferred thresholds including 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%. Comparisons are typically performed over defined regions to ensure maximum alignment and correspondence within a specified comparison window. These comparisons can be performed using BLAST or BLAST 2.0 sequence comparison algorithms with preset parameters or through manual alignment and visual inspection. Sequences that meet these criteria are described as "substantially identical." This definition also applies to complementary sequences of the test sequence and covers sequences that may have deletions, additions, or substitutions. Better algorithms for such comparisons can adapt to vacancies and similar variations.

The terms "treat," "treating," and "treatment" are used interchangeably and refer to actions that partially or completely prevent, alleviate, reduce, or control symptoms, secondary conditions, or illnesses associated with cancer. Specifically, "treatment" refers to the application or administration of the material composition of the pharmaceutical compound described herein to an individual experiencing symptoms, secondary conditions, or illnesses associated with cancer. The goal is to partially or completely alleviate or reduce one or more symptoms, secondary conditions, or illnesses, delay their onset, inhibit their progression, reduce their severity, or reduce their incidence. Treatment may also be provided to individuals who only exhibit early signs of such symptoms, conditions, or disease to reduce the risk of developing other complications of the disease. Treatment is generally considered "effective" if it reduces one or more symptoms or clinical indicators (as defined in this document). Alternatively, treatment is considered "effective" if it slows or halts the progression of symptoms, conditions, or disease.

The term "BBB" refers to any blood-brain barrier present in the brain and central nervous system.

When referring to nucleic acids or proteins, the term "isolated," as used herein, means that the nucleic acid or protein is essentially free from other cellular components typically associated with it in its natural environment. It can exist in a homogeneous form and can be in a dry or aqueous solution. The purity and homogeneity of the isolated material are typically assessed using analytical chemistry methods such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. Proteins that are major components in formulations are considered substantially purified.

As used herein, the term "approximately" refers to a range of values that covers the specified value and includes a range of values that are reasonably similar to those generally skilled in the art. For example, "approximately" is defined as within one standard deviation of a measurement based on widely accepted measurements in the industry. Additionally, "approximately" can extend to a range of ±10% of the specified value.

Treatment of brain cancer remains challenging, partly due to the biological nature of the BBB. Aptamers are considered small oligonucleotides that can be used as antibody alternatives. Compared to monoclonal antibodies, aptamers are small in size, thus offering the advantage of targeted delivery to specific tissue compartments (e.g., the CNS). Recent studies have shown the potential of aptamers in neurooncology and neurodegenerative diseases ( Murakami, K., Izuo, N., Bitan, G. Aptamers targeting amyloidogenic proteins and their emerging role in neurodegenerative diseases. J Biol Chem 298, 101478 (2022) ). However, no aptamer therapies have been reported for use in brain cancer.

Therefore, this disclosure provides a novel DNA therapy. Specifically, it provides an aptamer comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1 to 4, or a variant thereof, or a salt thereof. The aptamer is characterized by being a nucleic acid molecule capable of binding to a target molecule with high specificity and affinity. Almost all aptamers are non-naturally occurring molecules. The aptamer possesses several advantageous features for targeted drug delivery, including ease of selection and synthesis, high binding affinity and specificity, low immunogenicity, and general synthetic accessibility. It can effectively deliver anticancer agents (such as chemotherapy drugs, toxins, and siRNA) to cancer cells in vitro.

Aptamers can be selected in vitro from a broad library of randomized sequences using a method called systematic evolution of ligands by exponential enrichment (SELEX). Alternatively, slow dissociation rate modified aptamers (SOMAmers) can be developed. The application of SELEX and SOMAmer techniques involves incorporating functional groups that mimic the amino acid side chains, thereby enhancing the chemical diversity of aptamers. This method allows for the enrichment and identification of high-affinity aptamers that are specific to proteins.

Aptamers can be produced via synthetic methods using techniques familiar to those skilled in the art. An example is chemical synthesis on a solid support, typically employing phosphamide chemistry. In this process, the solid-supported nucleotide undergoes detrimethylation and is subsequently coupled with an appropriately activated nucleoside phosphamide to form a phosphite triester bond. This is followed by an end-capping step and oxidation of the phosphite triester using an oxidizing agent. This cycle is repeated to construct the aptamer.

One example of a DNA therapy is AptBCis1. AptBCis1 is a cisplatin-binding, BBB-penetrating, and lung cancer-targeting DNA aptamer. Its scaffold, AptBCis1, was identified in vivo via SELEX using a mouse model of orthotopic lung cancer (LM). AptBCis1 binds to EAAT2, nucleolin, and YB-1 proteins. These interactions may contribute to AptBCis1's BBB penetration, lung cancer targeting, nuclear translocation, and cisplatin-release characteristics. Data showed that AptBCis1 exhibited promising tumor-suppressive activity at lower cisplatin concentrations in both orthotopic and subcutaneous xenograft mouse models of lung cancer. These results demonstrate the translational potential of AptBCis1 in lung cancer (LM) and in cancers where platinum-based chemotherapy is the standard treatment. For example, this disclosure demonstrates the systemic anticancer effect of the drug because the new treatment effectively inhibits cancer growth at lower platinum equivalent concentrations (xenograft mouse model).

The aptamers described in this disclosure can be characterized by various chemical modifications, including substitutions at the sugar, phosphate, and/or base positions of nucleic acids. Such modifications can improve the stability of the aptamer or make it more resistant to degradation. Examples of such modifications include incorporation of modified nucleotides, addition of end-capping moieties (such as 3' capping), binding to high molecular weight non-immunogenic compounds (such as polyethylene glycol (PEG)), linking to lipophilic compounds, and alteration of the phosphate backbone.

The polynucleotide variants disclosed herein may exhibit at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% primary sequence identity with one of SEQ ID NO 1 to 8.

Modifications that produce a population of nucleotide variants resistant to nucleases may include one or more substituted nucleotide internucleotide bonds, modified sugars, modified bases, or combinations thereof. Such modifications include, but are not limited to, 2'-sugar modifications, 5-pyrimidine modifications, 8-purine modifications, modifications at the exocyclic amine site, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodouracil; skeletal modifications, thiophosphate or alkylphosphate modifications, methylation, and unusual base pairings, such as isobasic isocytidine and isoguanidine. Modifications may also include 3' and 5' modifications, such as end capping.

In other embodiments, the nucleotide comprises a modified glycosyl group, such as one or more hydroxyl groups halogenated, aliphatic groups replaced, or functionalized into an ether or amine. Other modifications are known to those generally skilled in the art.

This disclosure also includes pharmaceutical compositions containing the adaptor or adaptor therapy described herein. In some embodiments, the composition includes an effective amount of the adaptor or adaptor therapy, alone or in combination with one or more pharmaceutically acceptable carriers.

The aptamers described herein can form complexes with therapeutic agents. Preferably, the therapeutic agent is a drug associated with CNS diseases such as CNS cancers, neurodegenerative diseases, or diseases related to CNS damage. As previously mentioned, the aptamers described herein and their various embodiments can be used to transfer portions or compounds (such as therapeutic agents or imaging agents) into cells.

This disclosure also provides a method for treating a disease. The method involves administering an effective amount of the compound described herein to an individual in need.

The conjugates described herein can be administered to individuals via various delivery methods (including oral administration, suppository administration, and topical application) and routes (such as intravenous, intraperitoneal, intramuscular, intralesional, intrathecal, intrasheath, intranasal, or subcutaneous administration). This also includes implanted slow-release devices, such as microosmotic pumps. Administration can be performed via any route, including non-enteric and transmucosal methods (e.g., intracheal, sublingual, palatine, gum, nasal, vaginal, rectal, or percutaneous).

Non-enterointestinal administration covers a variety of routes, including intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial routes. Additional delivery methods may include liposome formulations, intravenous infusion, percutaneous patches, etc. The term "co-administration" refers to the administration of the conjugate described herein simultaneously, before, or immediately after one or more other therapies (such as cancer treatments, such as chemotherapy, hormone therapy, radiation therapy, or immunotherapy). The conjugate disclosed herein can be administered alone or in combination with other treatments. Co-administration covers the simultaneous and sequential delivery of the conjugate, whether administered alone or in combination with other drugs.

The compounds described herein can be used to treat or prevent pathologies, such as diseases or conditions, or to alleviate symptoms of such diseases or conditions in patients. For example, the compounds described herein can be used to treat or prevent cancer or pathologies related to CNS diseases. Examples of CNS diseases include, but are not limited to, CNS tumors and neurodegenerative diseases, such as neurodegenerative diseases and diseases related to CNS damage (such as stroke).

Examples of CNS tumors include, but are not limited to, metastatic brain tumors, neuroblastomas, malignant lymphomas, germ cell tumors, meningiomas, pituitary adenomas, schwannomas, glioblastomas, malignant gliomas, diffuse or degenerative astrocytomas, oligodendrogliomas, or any other primary or secondary malignant tumors of the central nervous system.

Examples of drugs associated with CNS cancers include, but are not limited to, chemotherapy drugs, antibody drugs, and ADCs. Examples of drugs targeting CNS cancers include, but are not limited to, ABT-414 (depatuxizumab mafodotin), AMG-595 (an anti-EGFR ADC with DM1), anti-TfR ADCs with linked low-molecular-weight delivery systems, bispecific anti-TfR and anti-EGFR ADCs with low-molecular-weight effective payloads; trispecific mAbs targeting TfR, EGFR, and tumor-specific molecules; temozolomide (an alkylating agent), lomustine, procarbazine, nimustine (ACNU), vincristine, and bevacizumab. Monoclonal antibodies (mAbs) against vascular endothelial growth factor (VEGF), bis(chloroethylnitrosourea) (BCNU), BCNU powder tablets (alkylated slow-release agent), axitinib, tesevatinib, nintedanib, sunitinib, pazopanib, sorafenib, cabozantinib, vandetanib The following are listed: anib, motesanib, cediranib, regorafenib, tivozanib, linifanib, dasatinib, imatinib, quizartinib, vemurafenib, dabrafenib, and trametinib.

Examples of neurodegenerative diseases include, but are not limited to, neurodegenerative diseases, neuromuscular diseases (such as muscular atrophy, amyotrophic lateral sclerosis), brain diseases (such as attention deficit hyperactivity disorder (ADHD), Bell's palsy, carpal tunnel syndrome, cerebral aneurysm, diabetic neuropathy, epilepsy, migraine and headache syndrome, stroke and traumatic brain injury), and spinal diseases (such as spina bifida, spinal cord injury, and spinal muscular atrophy).

Examples of neurodegenerative diseases include, but are not limited to, Alzheimer's disease, ataxia, Huntington's disease, Parkinson's disease, motor neuron disease, Lewy body disease, spinal muscular atrophy, multiple systemic atrophy and progressive supranuclear palsy, mild cognitive impairment, migraine, multiple sclerosis, myasthenia gravis, or amyotrophic lateral sclerosis (ALS).

Examples of medications associated with neurological disorders include, but are not limited to, donepezil (aricept), galantamine (razadyne), rivastigmine (exelon), iduxitase beta, anti-TfR ADCs containing iduxuronate-2-sulfatase, bispecific RmAb158-scFv8D3, anti-TfR mAb containing erythropoietin, scFab containing anti-TfR mAb and anti-Aβ mAb31, aducanumab, levetiracetam (Keppra), topiramate (Topamax), lamotrigine (Lamictal), oxcarbazepine (Trileptal), sodium valproate (Depakote), and gabapentin. Neurontin, pregabalin (Lyrica), carbidopa, levodopa, pramipexole (Mirapex), ropinirole (Requip), rotigotine (Neupro), clozapine, quetiapine, olanzapine, propranolol, alteplase (t-PA), reteplase, urokinase, prourokinase, tenecteplase, anistreplase, and streptococcal kinase.

Patients or individuals with pathological conditions, that is, patients or individuals treated by the method of this invention, can be vertebrates, more specifically mammals, or more specifically humans.

In light of this disclosure, the preparation of pharmaceutical compositions will be known to those skilled in the art. Typically, such compositions can be prepared as injectable preparations, in the form of liquid solutions or suspensions; in solid forms suitable for dissolving or suspending in a liquid prior to injection; tablets or other solids for oral administration; delayed-release capsules; or any other form currently in use, including eye drops, creams, lotions, ointments, inhalers, and the like. Surgeons, physicians, or healthcare professionals may also find it particularly useful to use sterile formulations (such as saline-based washes) to treat specific areas within a surgical site.

All disclosures and patents cited herein are incorporated herein by reference as if specifically and individually incorporated herein by reference. The citation of disclosures and patents does not imply acceptance that any disclosure or patent is prior art, nor does it constitute any acceptance of its content or date. This disclosure has now been described in writing, and those skilled in the art will recognize that the invention can be practiced in various embodiments, and that the foregoing description and the examples below are for illustrative purposes and not for limiting the scope of the following patent application.

Implementation Examples

Example 1. An isolated aptamer comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:1 to 8, or a variant thereof, or a salt thereof.

Example 2. An isolated aptamer as in Example 1, wherein the polynucleotide sequence of SEQ ID NO: 1 to 8 or a variant thereof or a salt thereof has a G-tetramer structure.

Example 3. An isolated aptamer as described in any of Examples 1 to 2, wherein the aptamer comprises a polynucleotide sequence of SEQ ID NO: 1, 3, 5, or 7, or a variant thereof, or a salt thereof.

Example 4. An isolated aptamer as described in any of Examples 1 to 3, wherein the aptamer comprises a polynucleotide sequence of SEQ ID NO: 1 or 5 or a variant thereof or a salt thereof.

Example 5. An isolated aptamer of any of Examples 1 to 4, wherein the polynucleotide sequence of SEQ ID NO: 1 or 5 or a variant thereof or a salt thereof forms 3 stem loops having a total of 9 GC pairs.

Example 6. A method for delivering a therapeutic agent to penetrate the BBB of an individual, comprising administering to the individual an adaptor-therapeutic agent complex as described herein, wherein the adaptor-therapeutic agent complex is a complex of one or more therapeutic agents and one or more adaptors as described in Examples 1 to 5.

Example 7. The method of Example 6, wherein the aptamer has a G-tetramer structure and comprises a polynucleotide sequence having fewer than 150 base pairs.

Example 8. The method of any of Examples 6 and 7, wherein the aptamer comprises a polynucleotide sequence having fewer than 100 base pairs or having about 50 to 100 base pairs.

Example 9. The method of any one of Examples 6 to 8, wherein the aptamer used in the method comprises a polynucleotide having the sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or 8 or a variant thereof or a salt thereof.

Example 10. The method of any one of Examples 6 to 9, wherein the aptamer used in the method comprises a polynucleotide having the sequence of SEQ ID NO: 1 or 3 or a variant thereof or a salt thereof.

Example 11. The method of any of Examples 6 to 9, wherein the aptamer used in the method comprises a polynucleotide having the sequence of SEQ ID NO:1 or 5 or a variant thereof or a salt thereof.

Example 12. An aptamer-therapeutic compound comprising a therapeutic agent that binds to an aptamer as described in any of Examples 1 to 5.

Example 13. An adaptor-therapeutic compound such as in Example 12, wherein the therapeutic is a drug associated with CNS diseases.

Example 14. An adaptor-therapeutic combination such as in Example 13, wherein the CNS disease is a CNS tumor or neurodegenerative disease, such as neurodegenerative diseases and diseases related to CNS damage (such as stroke).

Example 15. An adaptor-therapeutic combination as described in any of Examples 12 and 13, wherein the therapeutic is an anticancer agent or a drug for neurodegenerative diseases or CNS damage.

Example 16. An adaptor-therapeutic compound such as in Example 15, wherein the anticancer agent is a chemotherapy drug or a targeted cancer drug.

Example 17. A method for treating or preventing diseases in the CNS, comprising administering to an individual an adaptor-treatment combination as described in any of Examples 12 to 16, wherein the treatment is a drug for treating CNS diseases.

Example 18. A method for treating CNS cancer, comprising administering to an individual an adaptor-treatment combination as described in any one of Examples 12 to 16, wherein the treatment is an anticancer agent.

Example 19. The method of Example 18, wherein the CNS cancer is brain cancer, brain metastasis, degenerative astrocytoma, neuroglioblastoma or pia materoma.

Example 20. The method of Example 19, wherein the pia materomatosis is lung cancer pia materomatosis (LM).

Example 21. A method for treating or preventing neuropathic conditions, comprising administering to an individual an adaptor-therapeutic compound as described in any of Examples 12 to 16, wherein the therapeutic agent is a drug for treating neuropathic conditions.

Example 22. The method of Example 21, wherein the neurodegenerative disease is a neurodegenerative disease, a neuromuscular disease (such as muscular atrophy, amyotrophic lateral sclerosis), a brain disease (such as attention deficit hyperactivity disorder (ADHD), Bell's palsy, carpal tunnel syndrome, cerebral aneurysm, diabetic neuropathy, epilepsy, migraine and headache syndromes, stroke and traumatic brain injury), and a spinal disease (such as spina bifida, spinal cord injury, and spinal muscular atrophy). Example   

Materials and Methods   

Animal Research   

All mouse experiments were conducted on age-matched (6 weeks; approximately 20 g) BALB/c nude mice. Mice were obtained from the National Laboratory Animal Center (Taipei, Taiwan). The experiments were approved by the Department of Animal Care, Institute of Biomedical Sciences, Academia Sinica, Taiwan (IACUC Approval No.: IBMS-CRC100-P02).

Cells and cell culture   

The human lung cancer cell line CL1-5 was generated in our laboratory[13], and the PC9 cells were obtained from a collaborating laboratory at National Taiwan University Hospital. CL1-5 and PC9 cells were cultured in RPMI-1640 medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 100 μg/mL Primocin™ (InvivoGen, USA). Cells used in the experiments were passaged no more than 10 times after thawing. The mouse endothelial cell line bEnd.3 was obtained from the Bioresource Collection and Research Center Taiwan and cultured in high glucose Dulbecco's modified Eagle's medium (Life Technologies, Grand Island, NY) supplemented with 10% FBS, HEPEs, 100 U/mL penicillin, and 100 μg/mL streptomycin. Stable transfected colonies were maintained in media supplemented with either 2 pg/mL inonomycin (InvivoGen) or 400 μg/mL G418 (InvivoGen). Mycotic testing was performed periodically using the PlasmoTest™ kit (InvivoGen).

Chemicals, oligonucleotides, siRNA and antibodies   

All chemicals were purchased from Sigma-Aldrich. The aptamer library or modified aptamer system was synthesized by Integrated DNA Technologies (Coralville, IA, USA) or Purigo Biotech. The synthesized single-stranded DNA library consisted of 80 nucleotide-long single-stranded DNA with 40 random sequences side-joined to the primer sequence 5'-ACGCTCGGATGCCACTACAG [N] ₄₀ CTCATGGACGTGCTGGTGAC (SEQ ID NO:9), n=a, t, g, c.

Anti-luciferase antibody (sc-74548), EAAT2 siRNA (sc-35256), nucleolin siRNA (sc-29230), and YB-1 siRNA (sc-38634) were purchased from Santa Cruz. YH2AX (9718), YB-1 (4202), and EAAT2 (20848) antibodies were purchased from Cell Signaling. Human EGFR (aa 746-750 deletion; EGFR del19) antibody (MAB8336) was purchased from R&D Systems. Nucleolin antibody (ab22758) was purchased from Abcam.

pia mater carcinoma (LM) Orthotopic mouse model   

Six-week-old BALB/c nude mice were used to establish an orthotopic LM mouse model. Stable cells used included PC9 or CL1-5 cells expressing luciferase. To generate orthotopic LM mouse models for in vivo SELEX or for AptBCis1 efficacy studies, 1 × ^10⁶ or 3 × ^10⁵ cells were resuspended in 10 μL PBS (137 mM NaCl, 2.7 mM KCl, 10 mM _{Na₂HPO₄, 2} mM _KH₂PO₄ , pH 7.4) and directly seeded into the large cisterns of anesthetized recipient mice. Tumor burden was monitored using an in vivo imaging system (IVIS, Xenogen Caliper IVIS spectrum, USA), and mouse body weight was measured daily.

Subcutaneous xenograft mouse model   

For the subcutaneous xenograft mouse model, 5 × ^10⁵ PC9 cells were suspended in 10 μL PBS and inoculated into the right abdomen of 6-week-old BALB/c nude mice. Tumor size and mouse weight were measured daily. The volume was calculated as follows: (length × ^width² ) × 0.5¹.

Immunohistochemical studies   

To ensure successful establishment of the LM orthotopic mouse model, mice were euthanized on day 6 post-tumor inoculation. Strong bioluminescence (BLI) signals were then detected at anatomical locations in the brain and spinal cord using IVIS. The organs were perfused via the heart with PBS and 4% paraformaldehyde, followed by organ isolation. The isolated brain and spinal cord were fixed overnight at 4°C in 4% paraformaldehyde. The tissues were then embedded in paraffin wax to form tissue blocks. For histological studies, tissue slides were rehydrated and blocked with PBS containing 10% normal goat serum, 2% BSA, and 0.2% Triton X-100 (PBST). To detect tumor cells, a luciferase antibody (sc-74548, Santa Cruz) was prepared using PBST containing 10% normal goat serum and 1% BSA. After incubation overnight at 4°C, the sample was washed and stained with an HRP-bound secondary antibody (PK-6102, Vector).

Immunofluorescence research   

Immunofluorescence (IF) studies were performed on tissue sections to detect LM tumor cells, aptamers, and several protein markers. After sacrifice, mice were perfused cardiacally with PBS and 4% paraformaldehyde, followed by organ isolation. Isolated brains were fixed overnight in 4% paraformaldehyde at 4°C and then transferred to a 30% sucrose bath at 4°C for 24 hours. Samples were embedded in OCT compounds and cryosectioned. For IF staining, samples were blocked with PBST containing 10% normal goat serum and 2% BSA. Hybridization with luciferase, EGFRdel19, γH2AX, nucleolin, or YB-1 antibodies was performed overnight at 4°C. Secondary antibodies used included anti-FITC and anti-Cy3. Images were acquired using a Zeiss LSM700 confocal microscope (Carl Zeiss Microimage, Thornwood, NY).

In vivo SELEX   

For in vivo SELEX, 2 × ^10¹⁵ single-stranded DNA (ssDNA) oligonucleotides were dissolved in SELEX buffer (140 mM NaCl, 4 mM KCl, ₁ mM MgCl₂, ₁ mM CaCl₂, and 40 mM HEPES, pH 7.4). The ssDNA oligonucleotide library was intraperitoneally injected into LM mice. On day 6 post-tumor inoculation, tumor signals were detected at anatomical locations in the brain using IVIS (Xenogen Caliper IVIS spectrum, USA). Six hours after ssDNA library administration, mice were anesthetized and perfused with SELEX buffer to remove unbound oligonucleotides. The collected brain tissue was rapidly frozen in liquid nitrogen and then digested overnight at 55°C with proteinase K. DNA was extracted using a Gentra Puregene Tissue kit (Qiagen); the extracted DNA was used as the sample for oligonucleotide sequence amplification. The amplified oligonucleotide sequences were then isolated and folded as previously described ( Lai, WY, Wang, JW, Huang, BT, Lin, EP, Yang, PC A novel TNF-a-targeting aptamer for TNF-a-mediated acute lung injury and acute liver failure. Theranostics 9, 1741-1751 (2019) ), and injected intraperitoneally into LM mice for subsequent SELEX rounds. After the fourth round of SELEX, the amplified oligonucleotide sequences were ligated into CloneJet vectors (Thermo Fisher) for colony PCR. The amplified sequences were identified using Sanger sequencing (ABI3730, Applied Biosciences). Based on the predictions of the QGRS Mapper (https://bioinformatics.ramapo.edu/QGRS/index.php), the identified aptamers are grouped according to the existence of possible G-tetramer structures.

Cerebrospinal fluid (CSF) and plasma collection   

Plasma was sampled via cardiac puncture. Immediately after sampling, the blood was centrifuged, and the supernatant was transferred to sterile Eppendorf tubes. CSF was then sampled surgically. In short, the mice were anesthetized; using a microscope and micromanipulator (M650, Wild Heerbrugg), the skin, subcutaneous tissue, and muscles were dissected from the back of the neck to expose the cisterna magna. CSF was collected by direct capillary puncture of the dura mater. The sample was stored at -80°C until use.

AptB1 and platinum concentration measurement   

The concentrations of CSF and plasma AptB1 were measured by quantitative PCR (qPCR). For quantification, a standard curve for AptB1 was established using 80-mer oligonucleotides: the concentration of the 80-mer oligonucleotides was continuously diluted from 100 PM to 0.01 PM, resulting in the formula Y = 0.2982X + 5.3802, R = 0.9997. The concentrations of AptB1 were then measured accordingly. The concentrations of platinum in CSF or plasma were determined by inductively coupled plasma mass spectrometry (ICP-MS) (Thermo Fisher Scientific).

fit - Cis-Platinum Combination   

Cisplatinum powder was purchased from Sigma and dissolved in double-distilled water ( _ddH₂O ) at a concentration of 1 mg/ml. The applicators were denatured at 95°C; prior to cisplatin binding, they were cooled at 4°C and folded again at 37°C in SELEX buffer. Binding was performed according to the protocol ( Zhu, G., Niu, G., Chen, X. Aptamer-Drug Conjugates. Bioconjug Chem 26, 2186-2197 (2015) ). Successful binding was confirmed by gel electrophoresis (16% PAA non-denaturing gel; monoacrylamide to diacrylamide ratio 19:1); signal visualization was performed using STAINS-ALL (Sigma-Aldrich). The platinum aptamer was further analyzed using inductively coupled plasma optical emission spectrometry (ICP-OES) (Varian 720-ES, Agilent Technologies) to measure the amount of platinum bound. The platinum aptamer was dissolved in SELEX buffer and stored at -20°C until use.

LM In the orthotopic mouse model AptBCisl handle   

AptBCis1 or cisplatin was administered via tail vein on days 2, 3, 5, 7, 9, and 11 after tumor cell inoculation. The dosage of AptBCis1 was 1 mg/kg (approximately equivalent to 0.35 mg/kg of cisplatin), and the dosage of cisplatin was 2 mg/kg. Tumor burden was monitored using IVIS (Xenogen Caliper IVIS spectrum, USA), and mouse body weight was measured daily.

In subcutaneous xenograft mouse models AptBCis1 handle   

On day 6 post-inoculation, BALB/c nude mice inoculated with PC9 cells (5 × 10⁵) on their flanks were divided into 6 groups, at which time the tumor volume reached approximately 30-40 mm³. The drugs administered were (1) SELEX buffer, (2) cisplatin 0.35 mg/kg, (3) cisplatin 2 mg/kg, (4) AptB1 1 mg/kg, (5) AptBCis1 1 mg/kg (approximately 0.35 mg/kg cisplatin), or (6) AptBCis1 2 mg/kg (approximately 0.7 mg/kg cisplatin), via the tail vein. Tumor volume and mouse weight were measured daily.

Suitable sediment   

Cells or homogenized mouse brains (Dounce Tissue Grinder; Wheaton) were prepared in a buffer containing the protease inhibitor Thermo Fisher (50 mM Tris pH 7.5, 150 mM NaCl, ₅ mM MgCl2, 1 mM EDTA, 5% glycerol, 1% NP-40). Biotin-labeled aptamers and streptavidin were bound in 1× SSC buffer at 4°C for 3 hours, followed by washing with the buffer. Biotin-labeled aptamers or streptavidin-Sepharose agarose beads (Amersham pharmacia) and cell lysate were incubated overnight at 4°C, and the samples were washed four times with detergent-free buffer. The aptamer precipitate was then denatured in SDS sample buffer at 95°C and subjected to immunohistochemical analysis.

Nucleotide exonuclease analysis   

The control oligonucleotide 5'-FAM-TCGATCGGGGCGGGGCGATCG GGGCGGGGCGA (SEQ ID NO:10) (20 ng) or the 5'-FAM AptB1 aptamer (20 ng) was mixed with purified GST or GST-YB-1 protein (400 ng each) and incubated in a reaction buffer (50 mM Tris pH 7.5, 10 mM _MgCl2 ) at 35°C for 4 hours. After a brief centrifugation, the reaction products were separated using a 22% acrylamide gel containing 8M urea. The gel was then scanned using a Typhoon 9410 (GE).

Establishment of pia mater carcinoma (LM) Mouse models for in vivo use SELEX   

To identify adaptants that penetrate the BBB and target lung cancer, we established a mouse model of lung cancer in LM orthotopic presentation for in vivo SELEX. In short, stable CL1-5 lung cancer cells expressing luciferase were directly seeded into the large pit of mice (Figure 1a). Tumor burden was monitored daily using IVIS. Mice were sacrificed on day 6 post-inoculation, at which point strong bioluminescence (BLI) signals emitted from tumor cells were detected by IVIS at anatomical locations in the brain and spine (Figure 1b). The isolated brain and spinal cord were prepared into paraffin blocks and examined using anti-luciferase antibodies for H&E and immunohistochemical (IHC) staining. As shown in Figure 1C, tumor cells form tumor islands on the ventricular walls (small left image) and spine (small right image). The data indicate that a mouse model of lung cancer in the LM position was successfully established, which serves as a live in vivo SELEX platform for identifying adaptors that have penetrated the BBB.

Through living organism SELEX Identification AptB1 , A kind of penetration BBB and Targeted cancer adaptors   

For candidate aptamer identification, an oligonucleotide library containing ^10-15 80-nucleotide ssDNA molecules was intraperitoneally (IP) injected into LM mice. During each SELEX round, the brain was isolated 6 hours after ssDNA injection following perfusion with SELEX buffer, and the isolated brain was used for DNA extraction. The extracted DNA was amplified by PCR using specific primers designed for aptamer amplification. Amplicon was captured by beads and separated into single strands by heating. ssDNA was then injected again via IP to serve as the library for subsequent SELEX rounds. A total of 4 rounds of SELEX were performed, and the enriched oligonucleotide sequences were sequenced by Sanger sequencing (Figure 2A). Overall, nine candidate adaptors (AptB1 to AptB9) were identified (Table 1). Table 1. Adaptor sequences identified by SELEX in vivo.      

Nine candidate adaptors were amplified by PCR and labeled with the fluorophore anthocyanin-5 (Cy5). Based on QGRS Mapper predictions, these adaptors were divided into two groups: Group I (AptB1-4) contained a G-tetrameric structure, while Group II (AptB5-9) did not. The combined Group I or Group II adaptors were injected intraperitoneally (IP) into LM mice I or II, respectively. Significant BLI signals emitted from tumor cells were detected at anatomical locations in the brain. As shown in Figure 2B, strong Cy5 fluorescent signals emitted from anatomical locations in the brain and spine were detected by IVIS in LM mice I, exhibiting a gradual intensification pattern after IP injection, but were not detected in mice II. The data indicate that Group I adaptors have superior BBB penetration ability compared to Group II adaptors. LM mice were perfused with SELEX buffer and their brains were frozen into sections. As shown in Figure 2C, signals of group I adaptor (red) were detected in tumor cells (green) on the leptomeninges. The data further support the BBB-penetrating ability of group I adaptor and indicate its lung cancer targeting potential.

Next, the four adaptors Cy5-AptB1 to Cy5-AptB4 from Group I were individually IP-injected into LM mice, with the Cy5-labeled random sequence serving as a control. As shown in Figure 2D, Cy5 signals were detected from anatomical locations in the brain and/or spine of mice injected with AptB1 and AptB3. Since mice injected with AptB1 showed the most promising signal, frozen sections of the mouse brain were prepared for further confirmation. As expected, confocal microscopy images revealed AptB1 signals (pink) in tumor cells (green) on the pia mater, indicating its BBB penetration and lung cancer targeting ability (Figure 2E). Structural prediction using Mfolds indicated that AptB1 forms 3 stem-rings with a total of 9 GC pairs (Figure 2F).

To confirm the BBB penetration capability of AptB1, its intraperitoneal injection (IP) was administered to tumor-naive nude mice. Gradually increasing patterns of Cy5-AptB1 signals were detected in anatomical locations of the brain at 0.5, 1, 3, and 6 hours post-injection. The data further indicate that AptB1's BBB penetration capability is not related to BBB disruption caused by CNS cancer metastasis.

Detecting cerebrospinal fluid (CSF) Zhongzhi AptBl sequence   

To further confirm that the AptB1 signal detected by IVIS and confocal microscopy originated from aptamers located in the ventricular space rather than in the CNS microcirculation, CSF samples were collected directly from the cisterna magna using a glass capillary. In short, AptB1 was injected intravenously (IV) via the caudal vein, and CSF was collected approximately 30 minutes post-injection (Figures 3A-3C). To detect the AptB1 sequence, CSF was amplified by PCR using primers specific to our aptamer library. As shown by agarose gel electrophoresis, AptB1 amplicon was detected in both plasma and CSF samples from mice that had been given AptB1 (Figure 3D). Sequence accuracy was verified by Sanger sequencing (Figure 3E). Next, the percentage of BBB penetration was measured using qPCR. 20 μg of AptB1 or an 80-meric oligonucleotide random sequence (control) was injected intravenously via the tail vein. As previously mentioned, plasma and CSF were sampled approximately 30 minutes after drug administration, and the samples were quantified by qPCR. As shown in Figure 3F, the CSF/plasma ratio for AptB1 was 10.54%, and the CSF/plasma ratio for the oligonucleotide random sequence was 2.57%. These data further support the BBB penetration capability of AptB1.

AptBCis1 , An aptamer - Cis-platinum compounds in lung cancer LM It shows efficacy in diseases   

To develop an AptB1-chemotherapy conjugate for lung cancer (LM), we bound cisplatin to AptB1 to form AptBCis1. Successful binding was demonstrated on 16% non-denaturing polyacrylamide gel electrophoresis. AptBCis1 exhibited a slower migration rate due to the greater positive charge contribution from cisplatin (Figure 4A). Next, inductively coupled plasma optical emission spectroscopy (ICP-OES) analysis was performed on AptBCis1. The results showed that each AptB1 sequence segment contained approximately 27 platinum molecules.

We then used a lung cancer LM (Leydig-Leg) model to examine the tumor-suppressive effect of AptBCis1. In short, PC9 lung cancer cells were directly seeded into a large pool, and AptBCis1 (1 mg/kg, equivalent to 0.35 mg/kg cisplatin) or cisplatin (2 mg/kg) was administered via the tail vein IV on days 2, 3, 5, 7, 9, and 11 post-seedling (Figure 4B). Tumor growth was monitored by IVIS, and mouse body weight was measured daily. As shown in Figure 4C, the BLI signal emitted by cancer cells in the cisplatin-treated group was significantly stronger than that in the AptBCis1-treated group, indicating that the tumor-suppressive effect of AptBCis1 was superior to that of cisplatin. Consistent with IVIS data, on day 14 post-tumor inoculation, the weight loss in mice treated with AptBCis1 was moderate, while it was significant in the cisplatin-treated group (Figure 4D), supporting the tumor-suppressive effect of AptBCis1.

To confirm that the aforementioned tumor suppression was attributable to cisplatin-induced cytotoxicity, mice treated with AptBCis1 or cisplatin were sacrificed on day 8 post-tumor inoculation, and their brains were examined by immunofluorescence (Figure 4E). In short, frozen sections of the brain were co-stained with γH2AX-specific antibodies and luciferase or EGFR (aa746-750del)-specific antibodies. γH2AX represents a marker of cisplatin-induced DNA double-strand breaks ( Kinner, A., Wu, W., Staudt, C., Iliakis, G. Gamma-H2AX in recognition and signaling of DNA double-strand breaks in the context of chromatin. Nucleic Acids Res 36, 5678-5694 (2008) ); luciferase expression indicates tumor cells in our system, and EGFR (aa746-750del) antibodies specifically target PC9 cells expressing EGFR Del19. As shown in Figure 4F, tumor cells in the AptBCis1-treated group, highlighted by luciferase or EGFR signaling, exhibited depolymerization with fragmented cell clusters, indicating widespread cell death; conversely, tumor cells in the cisplatin-treated group possessed intact plasma membranes, indicating cell viability. Furthermore, γH2AX signaling was more extensively distributed in tumor cells in the AptBCis1-treated group than in the cisplatin-treated group, indicating a higher degree of DNA double-strand breaking induced by AptBCis1 treatment (Figures 4F-4G). In conclusion, our data suggest that AptBCis1 has a superior tumor-suppressive effect against LM in lung cancer compared to cisplatin.

AptBCis1 Inhibits lung cancer growth at lower cisplatin concentrations   

Data from LM mice showed that AptBCis1 had better tumor-suppressive activity at a lower equivalent systemic cisplatin concentration (0.35 mg/kg) compared to cisplatin (2 mg/kg). Therefore, determining the CSF platinum concentration in these two scenarios is particularly important.

In short, CSF was directly sampled from tumor-unaware mice 30 minutes after intravenous injection of 1 mg/kg AptBCis1 or 2 mg/kg cisplatin. Induction-coupled plasma spectrometry (ICP-MS) was performed on the CSF and paired plasma samples to determine platinum concentration. ICP-MS results showed that the platinum concentration in CSF treated with 1 mg/kg AptBCis1 was one-tenth that treated with cisplatin (2 mg/kg) (Figure 5A). Furthermore, ICP-MS data indicated that the CSF-to-plasma ratio was 10% and 20% when treated with AptBCis1 or cisplatin, respectively. In the AptBCis1 treatment group, better tumor inhibition was observed at lower CSF platinum concentrations. The data indicate that the antitumor effect of AptBCis1 exceeds the range of total platinum concentration and BBB penetration ability.

To verify this, we used a subcutaneous xenograft mouse model of lung cancer to examine the antitumor effect of AptBCis1. In short, PC9 lung cancer cells were seeded into the backs of BALB/c nude mice. On days 6, 7, 9, 11, 14, and 15 post-inoculation, mice were administered SELEX buffer via tail vein to control and different treatments: cisplatin 0.35 mg/kg, cisplatin 2 mg/kg, AptB1 1 mg/kg, AptBCis1 1 mg/kg (approximately 0.35 mg/kg cisplatin), and AptBCis1 2 mg/kg (approximately 0.7 mg/kg cisplatin) (Figure 5B). The results showed that AptBCis1 at 1 mg/kg (approximately 0.35 mg/kg cisplatin) had a greater tumor-suppressive effect than 0.35 mg/kg cisplatin, and its effect was only slightly lower than that of high-dose cisplatin (2 mg/kg). Notably, AptBCis1 at 2 mg/kg (approximately 0.7 mg/kg cisplatin) showed a similar tumor-suppressive effect to high-dose cisplatin (2 mg/kg) (Figures 5C-5D). Consistent with data from the LM orthotopic mouse model, results from the subcutaneous xenograft mouse model also showed that AptBCis1 exhibited better tumor-suppressive activity at lower equivalent cisplatin concentrations compared to cisplatin alone. The results further reinforced the assumption that the antitumor effect of AptBCis1 exceeded the total platinum concentration issue.

Furthermore, while neither 1 mg/kg nor 2 mg/kg of AptBCis1 resulted in weight loss, cisplatin 2 mg/kg caused approximately 10% weight loss during treatment (Figure 5E). These data indicate the toxicity of high doses of cisplatin and the relative safety of AptBCis1 at its effective doses.

AptB1 and EAAT2 Nucleolus and YB-1 protein binding   

To elucidate the mechanism, we then investigated AptB1 interacting proteins. AptB1 aptamer precipitation (AP, aptamer-based protein precipitation) was performed using total cell lysates prepared from mouse brain or PC9 lung cancer cells. Mass spectrometry (MS) analysis of the aptamer precipitation revealed three candidate proteins: excitatory amino acid transporter 2 (EAAT2), Y-box binding protein 1 (YB-1), and nucleolin (Figure 6A).

The results were then validated using AptB1 AP-immunomodulation. Immunomodulation was performed on mouse brain tissue and PC9 cell aptamer precipitates using EAAT2, YB-1, or nucleolin antibodies. Antibody specificity was confirmed by immunomodulation of total cell lysates, SiEAAT2, SiYB-1, or SiNucleolin, prepared from mouse endothelial cells (bEnd3) or human lung cancer (PC9) cells, with or without siRNA treatment. Results showed that EAAT2, YB-1, and nucleolin signals were detected in both mouse brain and PC9 aptamer precipitates, but with varying signal intensities. The EAAT2 signal was strongest in mouse brain but weakest in PC9. Nucleolin signaling was contrasting in both aptamer precipitates, being strongest in PC9 but weakest in the mouse brain (Fig. 6B). Confocal microscopy further visualized aptamer-protein interactions. As shown in Fig. 6C, the signals of AptB1 (red) and nucleolin (green, top inset) and AptB1 (red) and YB-1 (green, bottom inset) were co-localized (yellow) in PC9 cells (in both the cytoplasm and nucleus).

To further confirm the presence of AptB1 in the cell nucleus, PC9 cells treated with AptB1 were separated into cytosol and nucleus fractions. DNA was extracted independently from these two fractions, and the AptB1 sequence was amplified using an AptB1-specific primer. Successful amplification of AptB1 from the nucleus fraction indicated that AptB1 had entered the nucleus (Figure 6D).

Cisplatin interacts with DNA and forms covalent adducts with purine DNA bases and platinum compounds [6]. Therefore, the AptB1 DNA backbone must be digested before cisplatin can be released from AptBCis1 in order to exert its cytotoxic effect on target cancer cells. Previous studies have shown that YB-1 acts as an exonuclease ( Izumi, H., Imamura, T., Nagatani, G., Ise, T., Murakami, T., Uramoto, H. et al. , Y box-binding protein-1 binds preferentially to single-stranded nucleic acids and exhibits 3'T5'exonuclease activity. Nucleic Acids Res 29, 1200-1207 (2001) ). To test whether YB-1 functions as an exonuclease for AptB1, 5'-FAM-labeled AptB1 or control oligonucleotides were co-cultured with purified GST-YB-1 or GST protein. As shown in Figure 6F, both AptB1 and the control oligonucleotides were digested by GST-YB-1 protein, resulting in tailing on 22% polyacrylamide gel. This data supports the role of YB-1 as an exonuclease for AptBCis1.

In summary, we demonstrated that AptB1 binds to EAAT2, nucleolin, and YB-1. Binding to EAAT2 facilitates AptB1's BBB penetration. The interaction with nucleolin explains the efficient nuclear delivery of AptBCis1. Binding to YB-1 subsequently leads to the digestion of the AptB1 DNA backbone, promoting the release of cisplatin from AptBCis1. All these results collectively confirm the promising tumor-suppressive effect of AptBCis1 at lower cisplatin concentrations in both the orthotopic and subcutaneous xenograft mouse models of lung cancer (Figure 6G).

Figure 1 : Establishment of a mouse model of leptomeningeal carcinoma (LM) . (A) This workflow illustrates the establishment of an orthotopic LM mouse model by direct seeding of cells expressing luciferase into the cisterna magna. (B) Monitoring of mice by IVIS. Significant BLI signals were observed in the brain and spinal cord on day 6 post-tumor cell seeding, and the mice were sacrificed for IHC confirmation. (C) In the IHC study, tumor cells (red arrows) were observed in the ventricular space (left inset) and spinal cord (right inset).

Figure 2 : Identification of AptB1 , a BBB- penetrating and cancer-targeting adaptor, by in vivo SELEX . (A) The procedure illustrates the in vivo SELEX process. (B) Adaptors are grouped into Group I or Group II based on QGRS prediction. Group I adaptors contain a G-tetramer structure, while Group II does not. LM mice injected with Cy5-labeled Group I adaptors showed fluorescence signals in the brain/spinal cord in a progressively stronger pattern at 2 and 4 hours post-injection. (C) Confocal microscopy images showing Group I adaptor signals (red) in tumor cells (green) on the pia mater. (D) Strong Cy5 fluorescence signals were detected from the brain and spinal cord in LM mice injected with AptB1. (E) Confocal microscopy image showing AptBl signal (pink) in tumor cells (green) on the pia mater. (F) Mfold prediction of AptBl secondary structure.

Figure 3 : Detection of AptB1 in CSF . (A) CSF sample collected directly from the cisterna magna 30 minutes after intravenous injection of AptB1. (B) Gross image of the mouse cisterna magna (blue triangle). (C) CSF sampled from the cisterna magna using a capillary tube. (D) AptB1 amplification from CSF and plasma. (E) Confirmation of the accuracy of the amplified AptB1 sequence by Sanger sequencing. (F) Determination of AptB1 concentrations in CSF and plasma by qPCR. SD: Standard deviation.

Figure 4 : AptBCis1 , an aptamer - cisplatin conjugate , demonstrates efficacy in lung cancer LM disease. (A) Successful AptB1 and cisplatin conjugation shown on natural polyacrylamide gel. (B) This process plots the timeline of tumor cell inoculation and IV drug treatment (AptBCis1 or cisplatin). (C) Mice monitored by IVIS; BLI signal intensity indicates corresponding tumor burden. On day 14 post-tumor inoculation, mice in the cisplatin group showed a stronger BLI signal than mice in the AptBCis1 group (n=8 in each group). (D) Significant weight loss in the cisplatin group (** P < 0.01). Formula for weight change: (Day X / Day 2)%. (E) IVIS images were taken on days 2 and 8 post-tumor inoculation; cisplatin or AptBCisl was administered on days 2, 3, 5, and 7 post-tumor inoculation. Mice were sacrificed on day 8, and the brains were subjected to immunofluorescence studies. (F) Confocal microscopy images showed better preservation of tumor cell outlines in the cisplatin group (green; top inset: luciferase; bottom inset: EGFR) and a lower percentage of γH2AX-positive cells (red). (G) The percentage of γH2AX-positive cells was lower in the cisplatin group. Approximately 1200 cells and 450 cells were analyzed in the cisplatin and AptBCisl groups, respectively. An asterisk indicates a statistically significant difference. ** P < 0.01 (unpaired t- test). Data are mean ± SEM.

Figure 5 : AptBCis1 inhibits tumor growth at lower platinum concentrations. (A) Platinum concentrations in plasma and CSF were measured by ICP-MS. (B) This workflow illustrates the timeline of subcutaneous tumor cell inoculation and drug treatment in a mouse model of subcutaneous xenograft lung cancer. SELEX buffer, AptBl, AptBCis1, or cisplatin (n=4 per group) were administered via tail vein on days 6, 7, 9, 11, 13, and 15 post-tumor inoculation. (C) Tumor size was measured daily, and mice were sacrificed on day 23. (D) Gross image of the tumor. (E) Weight loss was more pronounced in the cisplatin 2 mg/kg group. An asterisk indicates statistical significance. * P < 0.05, ** P < 0.001 (non-paired t- test). Data are mean ± SEM.

Figure 6 : Interactions of AptB1 with EAAT2 , nucleolin, and YB-1 . (A) AptB1-AP/MS studies revealed three candidate AptB1 interacting proteins: EAAT2, YB-1, and nucleolin. (B) AP-immunomodulation confirmed the interaction between AptB1 and EAAT2, nucleolin, and YB-1 in PC9 cells and mouse brain. (C) Confocal microscopy images showing the colocalization (yellow) of AptB1 (red) and nucleolin (green, upper inset) or YB-1 (green, lower inset) in PC9 cells. (D) AptB1-treated cells were fractionated into cytosol and nucleus fractions. The AptB1 sequence was successfully amplified in both cellular fractions. (E) Purified GST and GST-YB-1 proteins confirmed by Coumarin staining and immunoblotting. (F) YB-1 exonuclease analysis results support the role of YB-1 as an exonuclease of AptB1. (G) The procedure illustrates the mechanism of the proposed AptBCis1 as a novel treatment for lung cancer with and without LM.

TW202600828A_114103678_SEQL.xml

Claims (22)

An isolated aptamer comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:1 to 8, or a variant thereof, or a salt thereof. The isolated aptamer of claim 1, wherein the polynucleotide sequence of SEQ ID NO: 1 to 8 or a variant thereof or a salt thereof has a G-tetramer structure. The isolated aptamer of claim 1 or 2, wherein the aptamer comprises a polynucleotide sequence of SEQ ID NO: 1, 3, 5 or 7 or a variant thereof or a salt thereof. The isolated aptamer of claim 1 or 2, wherein the aptamer comprises the polynucleotide sequence of SEQ ID NO: 1 or 5 or a variant thereof or a salt thereof. The isolated aptamer of claim 1 or 2, wherein the polynucleotide sequence of SEQ ID NO: 1 or 5 or its variant or salt of any of the foregoing forms 3 stem loops having a total of 9 GC pairs. In a method for delivering a therapeutic agent to penetrate the BBB of an individual, using an isolated aptamer of claim 1 or 2, the method comprises administering to the individual an aptamer-therapeutic agent complex as described herein, wherein the aptamer-therapeutic agent complex is a complex of one or more therapeutic agents and one or more aptamers as described in any of claims 1 or 2. The isolated aptamer of claim 6, wherein the aptamer has a G-tetramer structure and comprises a polynucleotide sequence having fewer than 150 base pairs. The isolated aptamer of claim 6, wherein the aptamer comprises a polynucleotide sequence having fewer than 100 base pairs or having about 50 to 100 base pairs. The isolated aptamer of claim 6, wherein the aptamer used in the method comprises a polynucleotide having the sequence of SEQ ID NO: 1, 2, 3 or 4 or a variant thereof or a salt thereof. The isolated aptamer of claim 6, wherein the aptamer used in the method comprises a polynucleotide having the sequence of SEQ ID NO: 1, 3, 5, or 7, or a variant thereof, or a salt thereof. The isolated aptamer of claim 6, wherein the aptamer used in the method comprises a polynucleotide having the sequence of SEQ ID NO:1 or 5 or a variant thereof or a salt thereof. An aptamer-therapeutic compound comprising a therapeutic agent that binds to an aptamer as claimed in any of claims 1 to 5. Such as the adaptor-therapeutic combination of claim 12, wherein the therapeutic is a drug associated with CNS diseases. For example, the adaptor-therapeutic combination of claim 13, wherein the CNS disease is a CNS tumor or neurodegenerative disease, such as neurodegenerative diseases and diseases related to CNS damage (such as stroke). Such as the adaptor-therapeutic combination of claim 12 or 13, wherein the therapeutic is an anticancer agent or a drug for neurodegenerative diseases or CNS damage. Such as the adaptor-therapeutic compound of claim 15, wherein the anticancer agent is a chemotherapy drug or a targeted cancer drug. In a method of treating or preventing diseases in the CNS, using an isolated adaptor of claim 1 or 2, the method comprises administering to an individual an adaptor-treatment combination of any one of claims 12 to 16, wherein the treatment is a drug for treating CNS diseases. In a method of treating CNS cancers, the isolated aptamer of claim 1 or 2 comprises administering to an individual an aptamer-treatment complex of any one of claims 12 to 16, wherein the treatment is an anticancer agent. For example, the isolated adaptor in claim 18, wherein the CNS cancer is brain cancer, brain metastasis, degenerative astrocytoma, neuroglioblastoma, or pia materoma. For example, the separated adaptor in claim 19, wherein the pia mater is lung cancer pia mater (LM). In a method of treating or preventing neuropathic conditions, such as the isolated adaptor of claim 1 or 2, the method comprises administering to an individual an adaptor-treatment combination of any one of claims 12 to 16, wherein the treatment is a drug for neuropathic conditions. For example, the fitting for separation in claim 21, wherein the neurodegenerative disease is a neurodegenerative disease, a neuromuscular disease (such as muscular atrophy, amyotrophic lateral sclerosis), a brain disease (such as attention deficit hyperactivity disorder (ADHD), Bell's palsy, carpal tunnel syndrome, cerebral aneurysm, diabetic neuropathy, epilepsy, migraine and headache syndrome, stroke and traumatic brain injury) and a spinal disease (such as spina bifida, spinal cord injury and spinal muscular atrophy).