Scaffold

Technical Field

This invention relates to a novel method for trapping magnetic nanoparticles inside cancer cells. It could be applied in the treatment of cancer by magnetic field hyperthermia.

Background Art

Most current cancer treatments fail to achieve complete regression of tumor giving the first choice for cancer treatment to surgery, as it removes the main bulk of tumor. Target selection and drug resistance are the main challenges in cancer therapy till now.

The limitation of conventional cancer treatments to reach complete regression is a result to the heterogeneity of cancer cells, even in the same tumor. Heterogeneity of cancer cells leads to the development of a

Darwinian selection process (weaker cancer cells die and stronger ones survive) after exposure to conventional therapeutics.

To overcome the heterogeneity of cancer cells, the drug used must exert its effect on a common target that present in all cancer cells. As cancer cells sustain uncontrolled growth and divide continuously, Cyclin

Bl, a mitotic marker which plays a vital role in the mitotic program, represents a suitable target.

Nucleic acids are known to exhibit a high degree of specificity to recognize its target. In this invention, we used nucleic acids to build scaffold(s) inside cancer cells that recognizes and trap functional magnetic nanoparticles inside cancer cells. Then, thermal ablation of cancer cells takes place after exposure of magnetic nanoparticle to an alternative magnetic field. Disclosure of the Invention

The invention relates to intracellular immobilization of Cyclin Bl mRNA in the inner surface of cell membrane and subsequent targeting with magnetic nanoparticles which are coated by specific ligands, oligonucleotides, specific for Cyclin B 1 mRNA.

Deposition of magnetic nanoparticles inside cancer cells takes place in two steps; establishment of a scaffold from Cyclin Bl mRNA and assembly of magnetic nanoparticles on this scaffold.

Cyclin Bl mRNA, like any other mRNA, is found in the cytoplasm after transcription to be translated and then degraded. This makes targeting of rriRNAs by magnetic nanoparticles useless as a result of its unfixed state.

The ability of membrane anchored proteins to keep association with plasma membranes relays on a post-translational process in which a lipid residue is added to its C-terminus. A terra peptide is located at this terminus and known as CAAX motif. It is recognized by certain enzymes inside cells which add the lipid residue to Cysteine amino acid. We used this phenomenon for selective deposition of magnetic nanoparticles inside cancer cells in two steps; Scaffold establishment and trapping of magnetic nanoparticles.

For scaffold establishment step; when an oligonucleotide sequence specific for Cyclin Bl mRNA is modified with CAAX tetrapeptide, it will be recognized by the cellular enzymes and will undergo lipidation inside cells leading to the indirect association of Cyclin Bl mRNA with the plasma membrane. On this way Cyclin Bl mRNA become immobilized and represents a scaffold that could be targeted easily.

As regard to the second step; magnetic nanoparticles are functionalized with ligands, oligonucleotide sequences specific for another region of Cyclin Bl mRNA, which act as hooks that link magnetic nanoparticles to the established scaffold. In turn, functional magnetic nanoparticles will be trapped inside cancer cells (Fig. 1).

The key factor for trapping magnetic nanoparticles inside cancer cells is the presence of Cyclin Bl mRNA on which two regions are targeted; one region will be targeted for fixation and scaffold establishment and the other will trap magnetic nanoparticles inside cancer cells. In addition, more than one region on Cyclin Bl mRNA could be targeted in order to establish a tight scaffold. Also, functionalization of magnetic nanoparticles may be done using different sequences specific for Cyclin Bl mRNA to ensure trapping.

In normal resting cells, there is no division and in turn there is no Cyclin Bl mRNA. Consequently, there will be no trapping for magnetic nanoparticles and they will be eliminated (Fig. 2).

After deposition of magnetic nanoparticles inside cancer cells, thermal ablation could be achieved by exposing magnetic nanopartilcles to an alternative magnetic field. In addition, the antisense strands down- regulate Cyclin Bl formation which is essential key for cell division, resulting in growth arrest.

One great advantage in this strategy is overcoming heterogeneity of cancer cells, because synthesis of Cyclin Bl is not restricted on a certain type, grade or stage of cancers making the strategy suitable for all type of cancers. Brief Description of Drawings

Figure (1) shows a diagrammatic representation for the invented method. Peptide-Oligo conjugate is used for scaffold establishment. Peptide portion of the conjugate undergo lipidation by the cellular enzymes and is responsible for membrane anchoring whereas oligo nucleotide portion is an antisense sequence specific for Cyclin B 1 mR A. Then, functional magnetic nanoparticle are recognized and trapped by the scaffold. Thus the presence of Cyclin Bl mRNA which is a common target between all cancer cells is the key factor for trapping magnetic nanoparticles. a, Lipid residue; b, Cyclin Bl mRNA; c, Antisense oligonucleotide; d, Magnetic nanoparticles; e, Linker between magnetic nanoparticles and the coating ligands (oligonucleotides); f, Linker between the antisense oligonucleotide and the lipid residue; g, Cell membrane.

Figure (2) shows a diagrammatic representation for the inability of magnetic nanoparticles to be fixed inside the cell in the absence of target (Cyclin Bl mRNA). a, Antisense oligonucleotide; b, Magnetic nanoparticles; c, Linker between magnetic nanoparticles and antisense oligonucleotide; d, Linker between the antisense oligonucleotide and a lipid residue; e: Lipid residue; f: Cell membrane.

Modes for Carrying out the Invention

Genetic mutations in Cyclin Bl mRNA that alter its sequence represent a challenge for both scaffold establishment and deposition of magnetic nanoparticles. In order to overcome this limitation, Multisequence targeting could be used. In multisequence targeting, different regions on the Cyclin Bl mRNA could be targeted with its antisense oligonucleotide sequences for scaffold establishment and coating of magnetic nanoparticles. On this way multi-targeting ensures that, if one targeted sequence in Cyclin Bl mRNA contains a genetic mutation, another region will be targeted and trapping of magnetic nanoparticles will take place.

Oligonucletides used in this invention, could be DNA, RNA or any homologue that can recognize a targeted sequence. In addition, oligonucleotides could be single or double stranded and modification of antisense strands for both scaffold establishment and for functionalization of magnetic nanoparticles may take place at the 5 ^Λ end or at the 3 ^Λ end. Oligonucleotide degradation by the action nucleases may represent a limitation for trapping magnetic nanoparticles. Stability of targeting ligands may be improved by using locked oligonucletides or siRNA. Locked nucleotides are nucleic acids homologues that can resist the action of nucleases thanks to chemical modification(s) that make them inaccessible to nucleases. Also, using siRNAs enhance the stability of targeting ligands because it enters the cell as double stranded sequences and then separated into single stranded sequences.

The direct modification of an antisense oligonucleotide sequence with a lipid residue could improve the reaction kinetics; i.e enhances speed of scaffold establishment and in rum magnetic nanoparticles are trapped rapidly; as there will be no need for the addition of the lipid residue by the cellular enzymes. However, modification of an antisense oligonucleotide sequence with CAAX peptide for immobilization of Cyclin B 1 mRNA is preferred than modification of that sequence with a lipid residue directly because it ensures an orientation of the scaffold to be anchored inside cell.

Another mode for carrying out this invention is targeting the protein product, by aptamers which are oligonucleotide ligands with a high degree of specificity for its target. In this case, two aptamers will be used; one aptamer will be modified with CAAX motif in order to catch Cyclin B 1 and fix it in the cell membrane, and the other aptamer will be used for functionalization of magnetic nanoparticles.

In this invention, ligands used for scaffold establishment are peptid- oligo conjugates in which the oligonucleotide portion recognizes its target, Cyclin Bl mRNA, in a high specific manner and the peptide portion is responsible for membrane anchoring. Peptide aptamers may be a good choice to design scaffold ligands. In this case there will be no need for the peptide-oligo conjugates which have difficulty in synthesis to some extent. On this way, the whole sequence of the scaffold ligands will be amino acids, may be termed anchoring peptide, containing the CAAX tetra peptide at its C-terminus. In addition, peptidomimics may be used to resist peptide degradation and give the anchoring peptide more stability.

Also, functionalization of magnetic nanoparticles is not restricted on oligonucleotide ligands or its homologues, but, peptide ligands or peptide- mimics or any other ligand could be used for coating magnetic nanoparticles if it has the ability to recognize the established scaffold.

The ability of magnetic nanoparticles to be trapped inside cancer cells in this invention depends on ligands located on its surface and the targeted scaffold. Adaptation of these ligands to be specific for other target enables magnetic nanoparticles to be deposited in cells containing that target. In turn, a wide variety of diseases could be treated using the invented method.

The present conventional treatments for viral diseases are in general targeting the protein synthesis machinery of the virus using chemical molecules to inhibit enzymes that are essential for virus replication, or by enhancement of immune detection (vaccination). So, targeting R As which are the key indicator for the virus activity, with magnetic nanoparticles could be more effective tool for treatment of viruses that can escape immune detection.

Generally, viruses are known to exhibit a high frequency of genetic mutations. This explains the inability of the immune system to recognize and detect a previously infected virus. Any genetically mutated virus represents a new virus for the immune system. Fortunately, most of these viruses contain a conserved region in its genome. These conserved regions, or any viral conserved transcripts, could be used for trapping magnetic nanoparticles inside infected cells as described in the invented method, scaffold establishment and magnetic nanoparticles trapping.

In the same way, magnetic nanoparticles could be deposited inside any type of bacteria by choosing the suitable target, like targeting a conserved region in a given transcript of Tuberculosis or any other bacterium.

Furthermore, optimization of targeting sequences make the strategy suitable for all types of fungi, like targeting a conserved region in a given transcript of a certain type of Tinea or another fungus.

Also choosing a suitable sequence, make the strategy suitable for all types of parasites, like targeting a conserved region in a given transcript of Plasmodium Malaria or any other parasite.

Actually, changing target make the strategy suitable for all types of organisms containing a conserved region in its genome, like targeting a conserved region in a given transcript of a certain type of Rickettsia or another class of microbes.

Sequence optimization makes the strategy suitable for some autoimmune diseases, like targeting a specific region, in a given transcript coding for a pathogenic antibody or receptor.

The invented strategy could be adapted to be diagnostic, by labeling the oligonucleotides with a contrast label for imaging the target.

Beyond magnetic nanoparticles, the invented method could be used for selective deposition of any drug or prodrug in a targeted cell. This would minimize doses and increase specificity of drugs to exert its effect on the targeted cell only, e.g selective trapping of photo or radio sensitizers inside targeted cells whatever it were a cancer or infected cell.

Magnetic nanoparticles may be also replaced by gold nanoparticles in order to achieve hyperthermia in the photodynamic therapy.

Industrial Applicability

This method could be applied in producing therapeutic or imaging agents for cancer. There is flexibility in choosing the type, sequence and modification(s) of the ligand for scaffold establishment and functionalization of magnetic nanoparticles.

Antisense oligonucleotides like DNA, RNA or homologues could be used for targeting mRNA. In addition, aptamers, DNA, RNA, peptide or homologues, could be used for targeting the protein product. Sequence of ligands is specific for any conserved target and multisequence targeting could be used also. Nucleic acid ligands and homologues could be modified at the 5' end or at the 3 end. Modifications could be magnetic nanoparticles, imaging agent, drug or prodrug. In addition, the described methodology could be adapted for treatment or imaging a wide range of diseases including viruses, bacteria, parasites and fungi.