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Patent Application Titled "Programmable Self-Assembled Nanostructures Based on Sidechain-Modified Pna for the Multivalent Display of Ligands"...

June 17, 2014



Patent Application Titled "Programmable Self-Assembled Nanostructures Based on Sidechain-Modified Pna for the Multivalent Display of Ligands" Published Online

By a News Reporter-Staff News Editor at Life Science Weekly -- According to news reporting originating from Washington, D.C., by NewsRx journalists, a patent application by the inventor Appella, Daniel H. (Rockville, MD), filed on May 11, 2011, was made available online on June 5, 2014 (see also Patents).

No assignee for this patent application has been made.

Reporters obtained the following quote from the background information supplied by the inventors: "Multivalent (or polyvalent) interactions refer to the simultaneous binding of multiple ligands on the surface of one molecular entity to multiple receptors on another. The strength and specificity of multivalent interactions depends on the cumulative effect of all the ligands and all the receptors involved in the process. Within a multivalent array, a single, isolated ligand-receptor interaction may actually be weak; however, the combined effect of multiple ligand-receptor interactions can be very strong. Such multivalent interactions occur throughout biology, and are important in numerous processes, such as those involving receptors at the surfaces of cells. For example, cell attachment, wound healing, and the immune response are basic examples where multivalent interactions are important. Therefore, multivalent interactions can be directly linked to cancer metastasis, blood clotting, and the generation of antibodies from a vaccination (see generally, Mammen et al., Angew. Chem. Int. Ed., 37:2754-94 (1998)).

"Mimicking multivalent interactions on a synthetic scaffold is challenging, especially when large numbers of ligands (such as 5 or more) need to be displayed. There are numerous synthetic scaffolds that have been developed, but there are significant limitations that remain. Ideally, a scaffold for the multivalent display of ligands should be easily manipulated to display anywhere from 1 up to about 200 ligands in a controlled manner. Well-defined synthetic scaffolds have been developed for the display of small numbers of ligands. Such systems are good because a single synthetic entity can be made and isolated, but it is rare that such systems display more than 5 ligands. Beyond this, well-defined synthetic scaffolds become very challenging to make. To study the multivalent effects of larger numbers of ligands, scientists rely on synthetic systems that are less well-defined and consist of mixtures. In this area, it is common to use polymers, dendrimers, proteins, and synthetic nanostructures (such as gold nanoparticles) as the synthetic scaffold to support larger numbers of ligands. Unfortunately, these larger systems are heterogeneous mixtures where the number of ligands per scaffold cannot be rigorously defined. In these cases, scientists determine an average number of ligands per scaffold or report the range of ligands per scaffold. Heterogeneous mixtures are often not acceptable by FDA standards for application as a therapeutic. As such, improved scaffolds are needed."

In addition to obtaining background information on this patent application, NewsRx editors also obtained the inventor's summary information for this patent application: "In some aspects, the invention concerns composition comprising: strands of polynucleotide and strands of PNA, each PNA strand comprising: (i) from 2 to 50 nucleobase subunits; and (ii) one or more gamma substituents; the PNA strands being complementary to at least a portion of at least some of the polynucleotide strands, and the molar ratio of PNA strands to polynucleotide strands being at least 1:1. In some preferred embodiments, the polynucleotide strands are DNA. Some DNAs comprise at least 120 nucleotide subunits. In some embodiments the DNAs are single stranded. In other embodiments, RNA is the preferred polynucleotide. In some embodiments the RNAs are single stranded. Some RNAs comprise at least 120 nucleotide subunits. Certain embodiments contain PNA with a backbone having at least one cyclopentyl residue.

"Some compositions of the invention have gamma substituents that are capable of binding to a receptor on the surface of a cell, binding to a cell surface molecule, or eliciting an immune response. In some embodiments, the molar ratio of PNA strands to polynucleotide strands is 2:1 to 10:1. In certain embodiments, the ratio is 3:1 to 7:1 or 4:1 to 6:1.

"Preferred gamma substituents, independently, include --R--NX.sup.1X.sup.2, where: R is a C.sub.1-C.sub.12 alkyl; X.sup.1 and X.sup.2 are, independently, H, biomolecules, fluorescent groups, metal ligands, Michael acceptors, azides, alkynes, or thiols; where at least one of X.sup.1 and X.sup.2 are other than H. In certain embodiments, X.sup.1 and X.sup.2 are, independently, H, biotin, fluorescein, thiazole orange, acridine, pyrene, Alexafluor Dyes, polypeptide, sugars (such as mannose or lactose), nucleic acid derivatives, oligonucleotides, RGD (Arg-Gly-Asp) or cyclic RGD. Additional groups and ligands that may be attached to the multivalent nano peptide nucleic acid (MNP) for multivalent display include, but are not limited to, cyclodextrins, porphyrins, polyhedral cage compounds containing boron, and the compositions depicted in FIG. 19 (such as, biotin, 1,4,7,10-tetraazacyclododecane-N,N',N'',N''-tetraacetic acid (DOTA), diethylene triamine pentaacetic acid (DTPA), a cryptand, a crown ether (12-crown-4,15-crown-5,18-crown-6, dibenzo-18-crown-6, and diaza-18-crown-6, or derivatives, for example), a pyridine-containing ligand, or calixarenes (such as calix[4]arenes, e.g., cone-4-tert-butylcalix[4]arenetetra(diethylamide), and calix[6]arenes). In some embodiments, derivatives of the aforementioned X.sup.1 and X.sup.2 groups and ligands may be utilized. In addition to gamma substituents, the N-terminal PNA residues of the compounds described herein can also include substituents (R2 and R3) on the nitrogens of the N-termini. Accordingly, a single PNA residue can have up to 3 substituents, one gamma substituent and two terminal substituents.

"In some embodiments the individual PNA residues described herein can have one substituent. In some instances this substituent will be conjugated to the gamma carbon of the PNA residue. In some embodiments this substituent will be conjugated to the terminal nitrogen of the PNA residue. A PNA residue of this nature will be an individual residue in some embodiments. However, it may also be one of multiple PNAs in a larger strand. In some embodiments the PNAs described herein can be complexed with DNA to form an MNP.

"In some embodiments the individual PNA residues described herein can have two substituents. In some instances at least one of the two substituents will be conjugated to the gamma carbon of the PNA residue. In some embodiments, at least one of the two substituents will be conjugated to a terminal nitrogen residue. In some instances at least one of the two substituents will be conjugated to the gamma carbon of the PNA residue and the other will be conjugated to a terminal nitrogen residue. A PNA residue of this nature will be an individual residue in some embodiments. However, it may also be one of multiple PNAs in a larger strand. In some embodiments the PNAs described herein can be complexed with DNA to form an MNP.

"In some embodiments the individual PNA residues described herein can have three substituents. In some instances at least one of the substituents will be conjugated to the gamma carbon of the PNA residue. In some embodiments, two substituents will be conjugated to a terminal nitrogen residue. In some instances at least one of the substituents will be conjugated to the gamma carbon of the PNA residue and the other two will be conjugated to a terminal nitrogen residue. A PNA residue of this nature will be an individual residue in some embodiments. However, it may also be one of multiple PNAs in a larger strand. In some embodiments the PNAs described herein can be complexed with DNA to form an MNP.

"The invention also concerns methods of treating or inhibiting a disease state in a mammal comprising administering to said mammal an effective amount of a compound described herein wherein at least some of the gamma substituents are selected to bind to a receptor on the surface of a cell associated with said disease state, to hinder the ability of a cell surface molecule to interact with a ligand that may trigger or prolong a disease state, or elicit an immune response. In some embodiments, the disease state is related to, independently, cancer; infectious diseases caused by HIV, influenza, rhinovirus, rotavirus, E. coli, anthrax or cholera; diabetes (type 2), Chagas disease, chronic inflammatory diseases, and autoimmune diseases (see generally, Hecht et al., Curr. Opin. in Chem. Biol., 13:354-59 (2009) and Mammen et al.).

"In yet another aspect, the invention concerns methods of forming nanostructure platforms comprising contacting polynucleotide with PNA strands, wherein said PNA strands comprise:

"(i) from 2 to 50 nucleobase subunits, and

"(ii) one or more gamma substituents;

"wherein the molar ratio of said PNA strands to said one or more polynucleotide strands is greater than 1:1 and said PNA strands are complementary to a portion of said polynucleotide strands. The PNA and DNA strands are as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

"FIG. 1 presents a general scheme where 2 MNPs (each with one gamma sidechain) bind to a complementary oligonucleotide. More than one gamma sidechain, however, may be attached to the MNP (in other examples 2, 3, and 5 sidechains are attached, and more are possible). By using a longer oligonucleotide, more than just 2 MNPs may be assembled for multivalent display. Variation in the number of gamma sidechains and the sequence of the oligonucleotide can be used to create well-defined multivalent arrays of ligands attached to the MNP so that anywhere from 1 to 200 copies of the ligand are displayed. The distance between adjacent MNPs on the oligonucleotide can also be varied by altering the oligonucleotide's sequence to include segments that are non-complementary to the MNP.

"FIG. 2 presents a general scheme where 2 different MNPs (each with a different nucleobase base sequence and each one with a gamma sidechain that has its own ligand) bind to a complementary oligonucleotide that has a sequence to recognize both MNP sequences. Again, more than one gamma sidechain may be attached to either MNP.

"FIG. 3 shows constructs one side chain (no peg)PNA-(no peg)RGD and one side chain (peg)PNA-(no peg)RGD.

"FIG. 4 shows constructs one side chain (peg)PNA-(peg)RGD and one side chain (long peg)PNA-(peg)RGD.

"FIG. 5 shows a N-terminal side chain PNA-RGD construct.

"FIG. 6 shows a two-side chain PNA-RGD construct.

"FIG. 7 shows a three-side chain PNA-RGD construct.

"FIG. 8 shows a three-side-chain PNA-poly mannose derivative.

"FIG. 9 shows a five-side-chain PNA-Lactose construct.

"FIG. 10 shows one side chain (no peg)PNA and one side chain (two peg linker)PNA constructs.

"FIG. 11 shows N-Terminal Side Chain PNA construct.

"FIG. 12 shows a two-side chain (two peg linkers) PNA construct.

"FIG. 13 shows three-side chain (two peg linkers) PNA and five-side-chain PNA constructs.

"FIG. 14 shows Tm data from PNAs with various gamma-sidechains.

"FIG. 15 shows IC.sub.50 data for various multivalent constructs.

"FIG. 16 presents a schematic for attaching an agonist for an adenosine receptor (A2A) to a PNA.

"FIG. 17 presents a schematic for attaching an agonist for an adenosine receptor (A2A) to a PNA.

"FIG. 18 presents a schematic for attaching an agonist for an adenosine receptor (A2A) to a PNA.

"FIG. 19 presents structures of representative groups and ligands that can be attached to the MNP for multivalent display.

"FIG. 20 shows metastatic tumor foci in mouse lungs for mice treated with either vehicle (negative control), monovalent cyclic RGD ligand, or an MNP conjugated to 15 cyclic RGD ligands.

"FIG. 21 provides a graphical illustration of the tumor foci shown in FIG. 21."

For more information, see this patent application: Appella, Daniel H. Programmable Self-Assembled Nanostructures Based on Sidechain-Modified Pna for the Multivalent Display of Ligands. Filed May 11, 2011 and posted June 5, 2014. Patent URL: http://appft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearch-adv.html&r=1654&p=34&f=G&l=50&d=PG01&S1=20140529.PD.&OS=PD/20140529&RS=PD/20140529

Keywords for this news article include: Patents, Nitrogen, DNA Research, Legal Issues, Cancer Vaccines.

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Source: Life Science Weekly


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