US 2010O136614A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/013.6614 A1 Luo et al. (43) Pub. Date: Jun. 3, 2010

(54) DENDRIMER-LIKE MODULAR DELIVERY Publication Classification VECTOR (51) Int. Cl. CI2P 2L/00 (2006.01) (76) Inventors: Dan Luo, Ithaca, NY (US); Yougen C08G 83/00 (2006.01) Li, Pasadena, CA (US) (52) U.S. Cl. ...…. 435/68.1: 525/54.2 (57) ABSTRACT Correspondence Address: WILSON, SONSINI, GOODRICH & ROSATI Various nucleic acid-based matrixes are provided, compris 650 PAGE MILL ROAD ing nucleic acid monomers as building blocks, as well as PALO ALTO, CA 94304-1050 (US) nucleic acids encoding proteins, so as to produce novel bio materials. The nucleic acids are used to form dendrimers that are useful as Supports, vectors, carriers or delivery vehicles (21) Appl. No.: 11/583,990 for a variety of compounds in biomedical and biotechnologi cal applications. In particular, the macromolecules may be (22) Filed: Oct. 18, 2006 used for the delivery of drugs, genetic material, imaging components or other functional molecule to which they can Related U.S. Application Data be conjugated. An additional feature of the macromolecules is their ability to be targeted for certain organs, tumors, or types (60) Provisional application No. 60/727.961, filed on Oct. of tissues. Methods of utilizing such biomaterials include 18, 2005. delivery of functional molecules to cells. Patent Application Publication Jun. 3, 2010 Sheet 1 of 13 US 2010/013.6614 A1

Patent Application Publication Jun. 3, 2010 Sheet 2 of 13 US 2010/013.6614 A1

Patent Application Publication Jun. 3, 2010 Sheet 4 of 13 US 2010/013.6614 A1

Patent Application Publication Jun. 3, 2010 Sheet 5 of 13 US 2010/013.6614 A1

Patent Application Publication Jun. 3, 2010 Sheet 6 of 13 US 2010/013.6614 A1

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FIG.7

SECR D NO:96

Patent Application Publication Jun. 3, 2010 Sheet 8 of 13 US 2010/013.6614 A1

FIG. 11 Patent Application Publication Jun. 3, 2010 Sheet 9 of 13 US 2010/013.6614 A1

FIG. 12

FG, 13

Patent Application Publication Jun. 3, 2010 Sheet 10 of 13 US 2010/013.6614 A1

FIG.15. J SEG ID NO:100

SEQD NO:97. P, GTGAGTAGTGA AATTGACTCATGGACTAs #élé SEO D NO:99

SEOQ ID NO:98 ? · DNA

FIG. 16 SEO ID NO: 104

*woravovouvogvlowaaoauvoiejewou'257 0LLLLLLLLLSLLLLLLLLLLLLLL

SEOQ ID NO: 103 <-R dumbbel-DNA.

SEOQ ID NO 102 FIG. 17

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SEO DI NO:2 ? Patent Application Publication Jun. 3, 2010 Sheet 11 of 13 US 2010/013.6614 A1

Plasmid DNA as CnC , !

Schema of viral and non-viral hybrid system Patent Application Publication Jun. 3, 2010 Sheet 12 of 13 US 2010/013.6614 A1

Patent Application Publication Jun. 3, 2010 Sheet 13 of 13 US 2010/013.6614 A1

FIG. 21.

Seberinë ,

BOTN-spacer-DNA. (Bio-5p-SP).

BioTN-spacer-DNA * GGCG---TCSACF-5° Yo-ONA . Step. Bio-5p-CCGG AAC3...... tggcor-TTGACT's ...

US 2010/013.6614 A1 Jun. 3, 2010

and DNA-mediated supramolecular structures (Taton et al., polynucleotide is complementary to at least a portion of the Journal of the American Chemical Society 122:6305-6306 third polynucleotide, and where the first, second, and third (2000)), DNA sensing via gold nanoparticles (Elghanian et polynucleotides are associated together to form a multimer, al., Science 277:1078-81 (1997)), Y-shape DNA molecules and at least one of the first, second and third polynucleotides (Eckardt et al., Nature 420:286 (2002)) and DNA patterning are linked to at least one bioactive agent. In some embodi via dip-pen nanolithography (Demers et al., Science 296. ments, the multimers are trimers that are Y-shape or T-shape. 1836-8 (2002)). However, the preceding prior art DNA-based In one embodiment, all the trimers are Y-shape. In another structures are are further limited to linear DNA. Linear DNA embodiment, the all the trimers are T-shape. In yet other was first used to construct an artificial nano-structure (Chenet embodiments, the trimers are Y- and T-shape. al., Nature 350,631 (1991)). Using “double crossover DNA (two crossovers connecting two helical domains), a variety of 0012. In other aspects of the invention, a multimer mol geometric objects, periodic arrays and nanoscale mechanical ecule comprises a first, a second, a third and a fourth poly devices have been constructed (Yan et al., Nature 415, 62 nucleotide, where at least a portion of the first polynucleotide (2002):Yan et al., Science 301, 1882 (2003); Seeman, Trends is complementary to at least a portion of the second poly Biochem Sci 30, 119 (2005); Pinto et al., Nano Lett 5, 2399 nucleotides, where at least a portion of the first polynucle (2005)). Recently Lin et al. used a linear DNA molecule as a otide is complementary to at least a portion of the fourth cross-linker to construct a thermal-stimulative polyacryla polynucleotide, where at least a portion of the third poly mide hydrogel, creating a DNA-polymer hybrid hydrogel nucleotide is complementary to at least a portion of second system (Lin et al., J Biomech Eng 126, 104 (2004)). polynucleotide and where at least a porting of the third poly 0009. However, dendrimer-like nucleic acid compositions nucleotide is complementary to at least a portion of the fourth have not been utilized to effect delivery of bioactive agents to polynucleotides, and where at least one of the first, second, cells (either in a targeted or nonspecific manner). Therefore third and fourth polynucleotides are linked to at least one there is a need for new biomaterials that have applications in bioactive agent. In some embodiments, the multimers are diverse areas of biotechnology and medicine, and which pro tetramers that are X-shape or dumbbell shape. For dumbbell vide more effecient modular delivery, sufficient release and shapes, the second polynucleotide comprises at least portions effective cellular/tissue targeting. The present invention pro vides compositions and methods that provide dendrimer-like that are complementary to the first, third and fourth nucle nucleic acid-based products useful in biotechnology and otides. Similarly, the fourth polynucleotide comprises at least medicine as modular delivery vectors for a multitidue of Some portions that are complementary to the first, second and compounds. third polynucleotides. In one embodiment, all the tetramers are X-shape. In another embodiment, the all the tetramers are SUMMARY OF THE INVENTION dumbbell-shape. In yet other embodiments, the tetramers are X- and dumbbell-shape. 0010 Certain aspects of the present invention provide a 0013. In some aspects of the invention also provide a multivalent vector capable of providing a plurality of attach method of making a nucleic acid assembly by associating at ment points for a plurality of the same or distinct bioactive least two mutlimers together. In some embodiments, the mul agents. Such bioactive agents include without limitation, timers so assembled are all of one shape (i.e., Y, T, X- or therapeutics (e.g., drugs, nucleic acids, Small organic mol dumbbell shape). In yet, other embodiments, the multimers so ecules, inorganic molecules), targeting or delivery moieties assembled are of one or more different shape. Such Y, T, X (e.g., signal peptides, nucleic acid condensing peptides, anti or dumbbell-shape molecules are building blocks which form bodies, one or more receptor/ligand or other binding pair an assembled structure. In some embodiments, a multimer members, biotin or nucleic acids), labeling/staining moieties building block comprises at least one polynucleotide having a (e.g., quantum dots, dyes, stains, selection markers), as well Sticky end. In other embodiments, a multimer comprises as Solid Substrates (e.g., agarose beads, magnetic beads, etc.). polynucleotides, each of which comprises a sticky end. Therefore, a key feature of a multivalent vector is that any number of different chemical/biochemical entities can be 0014. In some embodiments, a nucleic acid assembly is linked directly or indirectly to the multivalent vector. Den produced by associating a plurality of multimers together. In drimers as described herein provide a multivalent and/or Some embodiments, such associations produce Dendrimer monodisperse structure that provides multiple sites for addi Like-Nucleic Acid Molecules (DL-NAMs). In yet other tion of one or more molecules of interest, including without embodiments, DL-NAMs comprise at least some linear linker limitation bioactive agents, targeting agents, selection mark nucleic acid molecules. In various embodiments, DL-NAMs ers, antibiotics, detection signals/labels, drugs ora combina are comprised of a single shape or at least two different shape tion thereof. In various embodiments, such vectors can be building block molecules. utilized to deliver one or more bioactive agents to a cell or [0015] In certain embodiments, the DL-NAMs are pro animal. In other embodiments, such Vectors can also be uti duced in a controlled fashion, by adding multimer building lized in diagnostics by targeting specific cells related to dis blocks in Successive rounds to producea highly branched, ease (e.g., pathogens, cancer, etc.). Moreover, such multiva tree-shape DL-NAMs. In some embodiments, DL-NAMs are lent vectors are utilized in vivo as well as in vitro. produced that are either isotropic or anisotropic, providing 0011. In some aspects of the invention a composition com molecules that are linked to various other biochemical/ prises a multimer molecule, including a first, a second, and a chemical entities (e.g., therapeutics, targeting/delivery third polynucleotide, where at least a portion of the first agents, labeling/staining agents, binding pair members, etc.). polynucleotide is complementary to at least a portion of the 0016. In some embodiments at least on polynucleotide second polynucleotide, where at least a portion of the first forming a multimer is linked to a delivery or targeting agent polynucleotide is complementary to at least a portion of the that is a peptide, polypeptide, a cell receptor or a receptor third polynucleotide, where at least a portion of me second ligand. US 2010/013.6614 A1 Jun. 3, 2010

0017. In other embodiments, at least one polynucleotide cally designed sticky ends. The ligation was unidirectional. forming a trimer, whether the first, second or third polynucle FIG. 1C depicts the assembly of second generation den otides is linked to at least one bioactive agent. In yet other drimer-like DNA (G). G DNA was ligated with six embodiments, Y-DNAs. FIG. 1D depicts the assembly of third generation 0018. This procedure, based on using Y-DNA as building dendrimer-like DNA(G) and G. blocks, is simple and robust For example, the 4th generation 0026 FIG. 2A depicts an evaluation of Yo-DNA by arga of DL-DNA is close to being monodisperse, even without rose gel. Lanes 1, 2 and 3 are oligonucleotides Y.Y.Y., purification. In addition, both the Y-DNA and the DL-DNA respectively. Lanes 4, 5 and 6 correspond to the annealing nanoparticles are very stable. Furthermore, no self-ligated products of (Y, and Y), (Y, and Y), and (Yo, and Y). products were detected, which was commonly seen in other respectively. Lanes 7, 8 and 9 correspond to the stepwise types of design (Ma et al., Nucleic Acids Res 14:9745-53 annealing products of (YY, and Y), (YoaY and Y): (1986), which is hereby incorporated by reference in its and (Y, Y and Y), respectively. Lane 10 corresponds to entirety). This key improvement was due to the unique design the one-pot annealing product of (YY, and Y). FIG. 2B of end sequences. Thus, specifically designed polynucle depicts an evaluation of Y-DNA stability. Lane 1 represents otides can be combined to form Y-DNA, and specific combi freshly made Y-DNA and lane 2 represents the same Y-DNA nations of Y-DNAs can be combined to construct DL-DNA. stored at 4°C. for 30 days. Both Y-DNA and DL-DNA may be 3-dimensional, and may 0027 FIG. 3 depicts the characterization of the first gen contain branches. eration dendrimer-like DNA(Gi). FIG. 3A depicts an 0019. In additional embodiments, DL-NAMs are utilized example of sequences of GIDL-DNA, where a Y-shape is first to form dendrimer structures that can be monodisperse and formed by SEQ ID NOS: 22, 27 and 32, and subsequently multivalent. In such embodiments, a dendrimer can be com multiple of said Y-shapes are joined together. FIG. 3B is a posed of a single shaped multimer or two or more different schematic drawing of the denaturation strategy used to con shaped multimers. Dendrimers provide a multivalent and/or firm the GI DL-DNA structure. After GI DL-DNA denatur monodisperse structure that provides multiple sites for addi ation, six oligonucleotides were generated; three of these six tion of one or more molecules of interest, including bioactive oligonucleotides were new species with a unique length (90 agents, selection markers, antibiotics, detection signals/la bases). The remaining three were 30 bases. In FIG. 3C, lane 1 bels, drugs or a combination thereof. In various embodi is Y-DNA and lane 2 is GIDL-DNA. In FIG. 3D, lane 1 is a ments, such vectors can be utilized to deliver one or more molecular marker (oligonucleotide YO>). Lane 2 is GIDL bioactive agents to a cell or animal, including through the DNA without denaturing. Lanes 3 and 4 correspond to 0.25 membrane for the cell or nucleus. ug and 0.5 lug of the denatured G1 DL-DNA, respectively. 0020. In some embodiments, the targeting or delivery FIG.3E presents an evaluation of GI DL-DNA stability. G1 agent is a peptide selected from adenovirus core peptide, a on Lane 1 was freshly made. G1 on Lane 2 was the same as synthetic peptide, a DNA condensing peptide, a cell targeting that on Lane 1 but was stored for 45 days before gel electro peptide, an endosome disrupting peptide, a nuclear targeting phoresis. peptide, an influenza virus HA2 peptide, a simian immuno 0028 FIG. 4A is a schematic drawings of G DL-DNA deficiency virus gp32 peptide, an SV40 T-Ag peptide, a VP22 (left) and other higher generation DL-DNA (right). FIG. 4B peptide, Adno mu peptide, SV40 NILS peptide, a Tat peptide depicts an evaluation of higher generation DL-DNA forma such as HIV Tat or a Rev peptide. tion. Lanes 1, 2, 3, 4 and 5 correspond to G DL-DNA, Ofe 0021. In some embodiments, the multimer building blocks DL-DNA, G DL-DNA, G DL-DNA and G DL-DNA, are ligated together. respectively. 0022. In some embodiments, a delivery or targeting pep (0029 FIG. 5 presents images of DL-DNA: AFM images tide is linked to one or more multimer building block by a of G4 DL-DNA on mica surface using standard silicon tip linker molecule. Linker molecules include nucleic acid, pep (lower left) and single walled carbon nanotube (SWNT) tip tide or hybrid nucleic acid-peptide molecules. (top left and right), and TEM image of G4 DL-DNA (lower [0023] In some embodiments, the multivalent vector of the right). Scale bars correspond to 100 nm. invention is linked to at least one therapeutic nucleic acid 0030 FIG. 6 depicts divergent and convergent synthesis of molecule, which includes a DNA vaccine, atherapeutic gene, a nucleic acid assembly. an RNAi, an siRNA, an aptame or an antisense molecule. 0031 FIG. 7 depicts dendrimer-like DNA. BRIEF DESCRIPTION OF THE FIGURES 0032 FIG. 8 depicts a trimer in accordance with the present invention (SEQID NOs: 22, 27 and 32). 0024. The illustrations included within this specification [0033] FIG.9 illustrates formation ofan X-shape molecule. describe many of the advantages and features of the invention. 0034 FIG. 10 illustrates joining of several X-shape mol It shall be understood that similar reference numerals and ecules. characters noted within the illustrations herein may designate the same or like features of the invention. The illustrations and 0035 FIG. 11 illustrates formation of a T-shape molecule. features depicted herein are not necessarily drawn to scale. 0036 FIG. 12 illustrates formation of several T-shape 0025 FIG. 1 is a schematic drawing of DNA molecular molecules into a dendrimer-like molecule. assembly. FIG. 1A depicts the assembly of Y-DNA. Three 0037 FIG. 13 illustrates formation of matrixes comprised oligonucleotides were annealed together to form one Y-shape of different shaped molecules. DNA, a basic building block for dendrimer-like DNA (Y- 0038 FIG. 14 illustrates X-Y-, T-DNA building blocks. Y+Y >Yo;Y,+Yp+Y >Y; Y.Y.Y. Y, Y 0039 FIG. 15 Shows an exemplary X-DNA (SEQ ID Y+Y->Y: Ya Ya Ya->Y). FIG. 1B depicts the NOs: 16 (i.e., 5" starting with CTGA . . . ), 17 (5" starting with assembly of first generation dendrimer-like DNA (G). The ACCT... ), 18 (5" starting with GAAT...) and 19 (5" starting core Yo-DNA was ligated with three “Y's, all with specifi with TCCG ...).

US 2010/013.6614 A1 Jun. 3, 2010 sequences may be used to prevent any significant effects due default parameters. The CLUSTAL program is well described to the information coding properties of nucleic acids, to elicit by Higgins et al., Gene, 73:237 (1988), Higgins et al., particular cellular responses or to govern the physical struc CABIOS, 5:151 (1989); Corpet et al., Nucl. Acids Res., ture of the molecule. A “nucleotide probe' or “probe' refers 16:10881 (1988); Huang et al., CABIOS, 8:155 (1992); and to a polynucleotide used for detecting or identifying its cor Pearson et al., Meth. Mol. Biol., 24:307 (1994), which are responding target polynucleotide in a hybridization reaction. hereby incorporated by reference in their entirety. The The nucleic acids may comprise intron and exon sequences, ALIGN program is based on the algorithm of Myers and modified sequences, RNA, DNA, or analogs thereof. Miller, supra. The BLAST programs of Altschul et al., JMB, 0054 As used herein, the terms “isolated and/or purified’ 215:403 (1990); Nucl. Acids Res., 25:3389 (1990), which are refer to in vitro preparation, isolation and/or purification of a hereby incorporated by reference in their entirety, are based nucleic acid molecule of the invention, so that it is not asso on the algorithm of Karlin and Altschul Supra. ciated with in vivo substances, or is substantially purified 0060 Software for performing BLAST analyses is pub from in vitro Substances. licly available through the National Center for Biotechnology 0055. The following terms are used to describe the Information (worldwideweb.ncbinlm.nih.gov). This algo sequence relationships between two or more polynucleotides: rithm involves first identifying high scoring sequence pairs (a) “reference sequence.” (b) “comparison window. (c) (HSPs) by identifying short words of length W in the query "sequence identity. (d) "percentage of sequence identity.” sequence, which either match or satisfy some positive-valued and (e) “substantial identity.” threshold score T when aligned with a word of the same 0056 (a) As used herein, “reference sequence' is a defined length in a database sequence. T is referred to as the neigh sequence used as a basis for sequence comparison. A refer borhood word score threshold. These initial neighborhood ence sequence may be a segment of or the entirety of a word hits act as seeds for initiating searches to find longer specified sequence. HSPs containing them. The word hits are then extended in 0057 (b) As used herein, "comparison window makes both directions along each sequence for as far as the cumu reference to a contiguous and specified segment of a poly lative alignment score can be increased. Cumulative scores nucleotide sequence, wherein the polynucleotide sequence in are calculated using, for nucleotide sequences, the parameters the comparison window may include additions or deletions M (reward score for a pair of matching residues; always >0) (i.e., gaps) compared to the reference sequence (which does and N (penalty score for mismatching residues, always <0). not include additions or deletions) for optimal alignment of For amino acid sequences, a scoring matrix is used to calcu the two sequences. Generally, the comparison window is at late the cumulative score. Extension of the word hits in each least 5, 10, or 20 contiguous nucleotides in length, and option direction are halted when the cumulative alignment score ally can be 30, 40, 50, 100, or longer. Those of skill in the art falls off by the quantity X from its maximum achieved value, understand that to avoid a high similarity to a reference the cumulative score goes to Zero or below due to the accu sequence due to inclusion of gaps in the polynucleotide mulation of one or more negative-scoring residue alignments, sequence, a gap penalty can be introduced and is subtracted or the end of either sequence is reached. from the number of matches. 0061. In addition to calculating percent sequence identity, 0058 Methods of alignment of sequences for comparison the BLAST algorithm also performs a statistical analysis of are well known in the art. Thus, the determination of percent the similarity between two sequences. One measure of simi identity between any two sequences can be accomplished larity provided by the BLAST algorithm is the smallest sum using a mathematical algorithm. Preferred, non-limiting probability (P(N)), which provides an indication of the prob examples of Such mathematical algorithms are the algorithm ability by which a match between two nucleotide or amino of Myers and Miller, CABIOS, 4:11 (1988), which is hereby acid sequences would occur by chance. For example, a test incorporated by reference in its entirety; the local homology nucleic acid sequence is considered similar to a reference algorithm of Smith et al., Adv. Appl. Math., 2:482 (1981), sequence if the Smallest sum probability in a comparison of which is hereby incorporated by reference in its entirety; the the test nucleic acid sequence to the reference nucleic acid homology alignment algorithm of Needleman and Wunsch, sequence is less than about 0.1, more preferably less than JMB, 48:443 (1970), which is hereby incorporated by refer about 0.01, and most preferably less than about 0.001. ence in its entirety; the search-for-similarity-method of Pear 0062. To obtain gapped alignments for comparison pur son and Lipman, Proc. Natl. Acad. Sci. USA, 85:2444 (1988), poses, Gapped BLAST (in BLAST 2.0) can be utilized as which is hereby incorporated by reference in its entirety; the described in Altschul et al., Nucleic Acids Res. 25:3389 algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA, (1997), which is hereby incorporated by reference in its 87:2264 (1990), which is hereby incorporated by reference in entirety. Alternatively, PSI-BLAST (in BLAST 2.0) can be its entirety; modified as in Karhn and Altschul, Proc. Natl. used to perform an iterated search that detects distant rela Acad. Sci. USA, 90:5873 (1993), which is hereby incorpo tionships between molecules. See Altschuletal. Supra. When rated by reference in its entirety. utilizing BLAST, Gapped BLAST, PSI-BLAST, the default 0059 Computer implementations of these mathematical parameters of the respective programs (e.g. BLASTN for algorithms can be utilized for comparison of sequences to nucleotide sequences, BLASTX for proteins) can be used. determine sequence identity. Such implementations include, The BLASTN program (for nucleotide sequences) uses as but are not limited to: CLUSTAL in the PC/Gene program defaults a wordlength (W) of 11, an expectation (E) of 10, a (available from Intelligenetics, Mountain View, Calif.); the cutoff of 100, M-5, N=-4, and a comparison of both strands. ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, For amino acid sequences, the BLASTP program uses as FASTA, and TFASTA in the Wisconsin Genetics Software defaults a wordlength (W) of 3, an expectation (E) of 10, and Package, Version 8 (available from Genetics Computer the BLOSUM62 scoring matrix. See worldwideweb.ncbi.n- Group (GCG), 575 Science Drive, Madison, Wis., USA). lm.nih.gov. Alignment may also be performed manually by Alignments using these programs can be performed using the inspection. US 2010/013.6614 A1 Jun. 3, 2010

0063 Comparison of nucleotide sequences for determina other understringent conditions (see below). Generally, Strin tion of percent sequence identity to the sequences disclosed gent conditions are selected to be about 5° C. lower than the herein can be made using the BlastN program (version 1.4.7 thermal melting point (T) for the specific sequence at a or later) with its default parameters or any equivalent pro defined ionic strength and pH. However, stringent conditions gram. By “equivalent program' is intended any sequence encompass temperatures in the range of about 1° C. to about comparison program that, for any two sequences in question, 20° C., depending upon the desired degree of stringency as generates an alignment having identical nucleotide or amino otherwise qualified herein. acid residue matches and an identical percent sequence iden 0068 For sequence comparison, typically one sequence tity when compared to the corresponding alignment gener acts as a reference sequence to which test sequences are ated by the preferred program. compared. When using a sequence comparison algorithm, 0064 (c) As used herein, “sequence identity” or “identity” test and reference sequences are input into a computer, Sub in the context of two nucleic acid sequences makes reference sequence coordinates are designated if necessary, and to a specified percentage of residues in the two sequences that sequence algorithm program parameters are designated. The are the same when aligned for maximum correspondence sequence comparison algorithm then calculates the percent over a specified comparison window, as measured by sequence identity for the test sequence(s) relative to the ref sequence comparison algorithms or by visual inspection. erence sequence, based on the designated program param When percentage of sequence identity is used in reference to eters. proteins it is recognized that residue positions which are not 0069. As noted above, another indication that two nucleic identical often differ by conservative amino acid substitu acid sequences are substantially identical is that the two mol tions, where amino acid residues are substituted for other ecules hybridize to each other under stringent conditions. The amino acid residues with similar chemical properties (e.g., phrase “hybridizing specifically to refers to the binding, charge or hydrophobicity) and, therefore, do not change the duplexing, or hybridizing of a molecule only to a particular functional properties of the molecule. When sequences differ nucleotide sequence under Stringent conditions when that in conservative Substitutions, the percent sequence identity sequence is present in a complex mixture (e.g., total cellular) may be adjusted upwards to correct for the conservative DNA or RNA. “Bind(s) substantially” refers to complemen nature of the substitution. Sequences that differ by such con tary hybridization between a probe nucleic acid and a target servative substitutions are said to have “sequence similarity” nucleic acid and embraces minor mismatches that can be or “similarity.” Means for making this adjustment are well accommodated by reducing the stringency of the hybridiza known to those of skill in the art. Typically this involves tion media to achieve the desired detection of the target scoring a conservative Substitution as a partial rather than a nucleic acid sequence. full mismatch, thereby increasing the percentage sequence (0070 “Hybridization” refers to a reaction in which one or identity. Thus, for example, where an identical amino acid is more polynucleotides react to form a complex that is stabi given a score of 1 and a non-conservative substitution is given lized via hydrogen bonding between the bases of the nucle a score of Zero, a conservative Substitution is given a score otide residues. The hydrogen bonding may occur by Watson between Zero and 1. The scoring of conservative substitutions Crick base pairing, Hoogstein binding, or in any other is calculated, e.g., as implemented in the program PC/GENE sequence-specific manner. The complex may comprise two (Intelligenetics, Mountain View, Calif.). Strands forming a duplex structure, three or more Strands 0065 (d) As used herein, “percentage of sequence iden forming a multi-stranded complex, a single self-hybridizing tity” means the value determined by comparing two optimally Strand, or any combination of these. A hybridization reaction aligned sequences over a comparison window, wherein the may constitute a step in a more extensive process, such as the portion of the polynucleotide sequence in the comparison initiation of a PCR, or the enzymatic cleavage of a polynucle window may include additions or deletions (i.e., gaps) as otide by a ribozyme. compared to the reference sequence (which does not include 0071. The term “hybridized as applied to a polynucle additions or deletions) for optimal alignment of the two otide refers to the ability of the polynucleotide to form a sequences. The percentage is calculated by determining the complex that is stabilized via hydrogen bonding between the number of positions at which the identical nucleic acid base or bases of the nucleotide residues. The hydrogen bonding may amino acid residue occurs in both sequences to yield the occur by Watson-Crick base pairing, Hoogstein binding, or in number of matched positions, dividing the number of any other sequence-specific manner. The complex may com matched positions by the total number of positions in the prise two strands forming a duplex structure, three or more window of comparison, and multiplying the result by 100 to Strands forming a multi-stranded complex, a single self-hy yield the percentage of sequence identity. bridizing strand, or any combination of these. The hybridiza 0066 (e)(i) The term “substantial identity” of polynucle tion reaction may constitute a step in a more extensive pro otide sequences means that a polynucleotide includes a cess, such as the initiation of a PCR reaction, or the enzymatic sequence that has at least 51%, 52%, 53%, 54%, 55%, 56%, cleavage of a polynucleotide by a ribozyme. 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 0072. As is known to one skilled in the art, hybridization 67%, 68%, or 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, can be performed under conditions of various stringency. 77%, 78%, or 79%, preferably at least 80%, 81%, 82%, 83%, Suitable hybridization conditions are such that the recogni 84%, 85%, 86%, 87%, 88%, or 89%, more preferably at least tion interaction between the probe and target ER-stress 90%, 91%, 92%, 93%, or 94%, and most preferably at least related gene is both sufficiently specific and sufficiently 95%,96%.97%.98%, or 99% sequence identity, compared to stable. Conditions that increase the Stringency of a hybridiza a reference sequence using one of the alignment programs tion reaction are widely known and published in the art. See, described using standard parameters. for example, (Sambrook, et al., (1989), supra; Nonradioactive 0067. Another indication that nucleotide sequences are In Situ Hybridization Application Manual, Boehringer Man substantially identical is if two molecules hybridize to each nheim, second edition). The hybridization assay can be

US 2010/013.6614 A1 Jun. 3, 2010

0082) Nucleic Acid Molecules of the Invention I0088 Additionally, any inorganic or organic molecules, 0083. In certain aspects, the nucleic acid molecules pro including amino acids, silicas, cytokines, such as interleu vide monomer building blocks and/or cross-linkers that form kins, biologics and drugs may be added to the nucleic acid a three-dimensional matrix or scaffold structure. A matrix of polymers to produce certain biological effects. Nucleic acids the invention can be comprised of nucleic acids that are provide a variety of molecular attachment sites and therefore X-shaped. Y-shaped T-shaped, dumbbell-shaped (e.g. FIGS. facilitate covalent, ionic and hydrogen bonding, as well as Van der Wals attachments, orother forms of attachment. 8-14) nucleic acids, or a combination thereof. Examples of I0089. In addition, the nucleic acids may be methylated, various shape nucleic acids (e.g., DNA) are disclosed in U.S. ethylated, alkylated, or otherwise modified along the back patent application Ser. Nos. 10/877.697 and 60/756,453, bone to influence degradation rates. Generally, methylated, which are incorporated by reference in their entirety. Nucleic hemi-methylated, ethylated, or alkylated nucleic acids will acid building blocks are utilized for produce DL-NAMs. In degrade more slowly. Other backbone modifications affect one embodiment, DL-NAMs are substantially comprised of ing degradation rates include the use of heteroatomic oligo Y-shape nucleic acids. In a further embodiment, the Y-shape nucleoside linkages as described in U.S. Pat. No. 5,677,437. nucleic acid is DNA. Additionally, modifications may be used to prevent the 0084. In other embodiments, DL-NAMs are formed of nucleic acid from being transcribed or translated in a given linear and branched nucleic acids. In yet further embodi tissue or organism. In addition, the nucleic acids may be ments, the linear or branched nucleic acids can be DNA, capped to prevent degradation. Such caps are generally RNA, PNA, TNA, LNA or any combination thereof. For located at or near the termini of the nucleic acid chains. example, a DL-NAMs comprise branched DNA that form Examples of capping procedures are included in U.S. Pat. building blocks Supporting the dendrimer structure and also Nos. 5.245.022 and 5,567.810. linking linear DNA that can, for example, be linked to a solid 0090. One aspect of the invention is directed to a matrix substrate. comprising nucleic acids that include X-shape, T-shape, 0085. In other embodiments, purified nucleic acids may be Y-shape or dumbbell-shape, which nucleic acids can be used linked to other nucleic acids or other compounds. Linking as building blocks for new, designer biomaterials. Thus the may be accomplished in a variety of ways, including hydro nucleic acid(s) have different shapes and one or more shapes gen bonds, ionic and covalent bonds, JC-TC bonds, polarization can be utilized as a monomer (e.g., building block) for con bonding, van der Wals forces. As used herein, “link’ and structing DL-NAMs. In one embodiment, branched nucleic “cross-link” are used interchangeably. More than one type of acids are all of one shape (X-, Y-, dumbbell- or T-shape), crosslinking may be used within a given biomaterial. Further which nuclei acids are used as monomers. In some embodi more, use of a type of crosslinking easily degraded in a cell ments, branched nucleic acids are prepared through the coupled with a more degradation resistant type of crosslink hybridization of the complimentary sequences of the pre ing may result in a biomaterial that is opened in two phases, designed oligonucleotides (Table 3). In some embodiments, one when the easily degraded crosslinks are broken and sec the nucleic acids are DNA, RNA, PNA, LNA or TNA. In ond when the more resistant crosslinks or the nucleic acid additional embodiments, one or more combinations of Such itself are degraded. Crosslinking may be accomplished by nucleic acids can be utilized as building blocks. In further UV radiation, esterification, hydrolysis, intercalating agents, embodiments, the monomers are linked to other monomers neoplastic agents, formaldehyde, formalin, or silica com by ligation. Therefore, the monomers can undergoea ligation pounds. Examples of linking include but are not limited to the reaction facilitated by a nucleic acid ligase. use of siloxane bridges as described in U.S. Pat. No. 5,214. 0091. Furthermore, the nucleic acids are capable of under 134. going enzymatic reactions. In some embodiments, the reac I0086 Crosslinking may occur between two strands of a tions include reactions by enzymes, wherein said one or more double stranded nucleic acid or between the strands of two enzyme is a DNA polymerase, RNA reverse transcriptase, separate double strands. It may also occur between two sepa terminal transferase, DNA ligase, RNA ligase, exonuclease, rate single strands. Double Strand to single strand crosslink ribonuclease, endonuclease, polynucleotide kinase, DNA ing is also possible, as is crosslinking between different methylase, or DNA ubiquitinase. Furthermore, reactions regions of one Strand. Linkers such as Small organic mol include any reaction wherein one or more enzyme is an ecules (esters, amines) or inorganic molecules (silicas, silox enzyme that shortens nucleic acids, lengthens nucleic acids, anes), including microparticles or nanoparticles thereof, may amplifies nucleic acids, labels nucleic acids, or a combination be used to attach bioactive agents to nucleic acids. Any of the of reactions/enzymes thereof. different shaped nucleic acids of the invention can be linked 0092 X-Shape or cross-linked by one or methods described herein. There 0093. In one aspect of the present invention, DL-NAMs fore, X-shaped, Y-shaped, T-shaped, dumbbell shaped or any are comprised entirely or at least in part of branched nucleic combination thereof can be linked to each other, as well as to acids that are X-shape nucleic acids. In one embodiment, the other chemical moieties or polymeric compounds. X-shape nucleic acid is DNA. In yet another embodiment, the 0087. In addition, in certain aspects, where nucleic acids matrix is comprised of X-shape DNA and/or RNA, or ana are linked to bioactive agents. Such bioactive agents can be logs/derivative thereof. In another embodiment, the matrix is selected as desired, including drugs, selection markers, comprised of X-shape DNA, and linear DNA, RNA or PNA. detectable signals, other therapeutic agents, peptides, such as In one preferred embodiment, the matrix is nearly entirely signal or cell targeting peptides, nucleic acid sequences, pro comprised of nucleic acids. In yet another embodiment, the teins (including antibodies), plasmids, viruses, viral vectors, X-shape nucleic acids are RNA. Small molecules, inorganic compounds, metals orderivatives 0094. In one embodiment, four different oligonucleotides thereof. Nucleic acids so linked can include antisense, with complimentary sequences, termed as XO1, X02, X03, siRNA, RNAi, aptamers or ribozymes. and X04 (Table 3), are hybridized with each other through an

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six free branches of G1), resulting in a second-generation TABLE 3 DL-DNA (G2). The third (G3), fourth (G4), and higher gen eration DL-DNA were assembled in a similar way. Note that Sequence Table the assembled DL-DNA (Gn) had only one possible confor SEQ ID NO Sequence mation due to the unidirectional ligation strategy. The general format of then'-generation DL-DNA is Gn=(Yo)(3Y)(6Y.) 16 3 º - TCGAAGGCTGATTCGGTTAGTCCATGAGTC-5 º . . . (3x2" 'Y), where n is the generation number and Yn is 17 3 º – AATTGACºTCATGGACºTATCATGCGGATCCA-5" the n' Y-DNA. The total number of Y-DNA in an nth-gen eration DL-DNA is 3x2"-2. The growth of DL-DNA from 18 3'-AGCTTGGATCCGCATGACATTCGCCGTAAG-5* n" generation to (n+1)" generation requires a total of 3x2" 19 3- GATCCTTACGGCGAATGACCGAATCAGCCT-5 new Y-DNA.a-l 0103) Three specific polynucleotides are combined to 2O 3'-TCGA-5 form each Y-DNA. First, the free energy (deltaG) was calcu 21 3 ' -AATT- S ' lated for a sequence. In general, a lower free energy is desired. However, intermediate-low deltaG arealso considered. Sec 22 3-AGCT-5 ond, the secondary, structure of the molecule is considered. In 23 3- GATC-5 general, the least amount of secondary structure is desired. Third, it needs to be determined if the molecule would forma 24 3-AGGCTGATTCGGT-5 self-dimer, as it should 5 not form a self-dimer. Fourth, me 25 3 " - GACT CATGGACTA- S " length is considered, which can vary depending on the design goals. The molecule should be long enough to form stable 26 3'-TGGATCCGCATGA-5 DNA structure. For Y-DNA, it should be more than 8 nucle otides (nt) long. Fifth, the helix geometry should be consid 27 3 " - CTTACGGCGAATG - S " ered. Half-turns should be considered as the quantum of DNA 28 3'-TAGTCCATGAGTC-s' nanostructure. The length between two junctions should be 5*n bp, where n is 0, 1, 2, 3 etc. Next, the G/C content should 29 3'-TCATGCGGATCCA-5." be considered, hione embodiment, sequences are chosen that 2O 3 * - CATTCGCCGTAAG-5* constitute about 50% G/C. Last, the symmetry of the mol ecule should be considered. Sequence symmetry (e.g., as 21 3'-ACCGAATCAGCCT-5 those occurring in Holliday junctions) of each arm should be avoided. For Y-DNA sequence design, all three oligonucle 0100 Thus, X-DNA can ligate with each other via T4 otides should be checked at the same time. In some embodi DNA ligase, resulting in highly branched dendrimer struc ments, for X-DNA, complementary segements/arms are ture. In some embodiments, linear nucleic acids, Y-shape, longer than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 T-shape, dumbbell-shape or dendrimer shape nucleic acids nucleotides. A number of programs are available online having the necessary Sticky ends can also be incorporated into which enable researchers to obtain detailed information branched dendrimer structure (e.g., DL-NAMs) formed of regarding a DNA sequence, Such as melting temperature, X-shape nucleic acids. Therefore, in Some embodiments, the self-priming, secondary structure formation, calculations of matrix is comprised of X-shape and one or more other shapes free energy and alternate structure form a given sequence. in a ratio of each monomer that is preselected as desired. Single stranded DNA folding software can also be used to check for complete complementarities of sequences and pro Y-Shape vide two-dimensional representations of the complex. Fur thermore, input branched DNA can be modified to model 0101. In another aspect, DL-NAMs are comprised of after multiple strand folding by sealing the double-stranded Y-shape nucleic acids. In one embodiment, the Y-shape open ends with a poly(NTP) hairpin or spacer. Examples of nucleic acid is DNA. In yet another embodiment, DL-NAMs Such sequence evaluation tools available online include: Y-shape DNA and/or RNA, or analogs/derivatives thereof. In Fisher Scientific at woldwideweb.firsheroligos.com/oligo another embodiment, DL-NAMs are comprised of Y-shape calconyl.asp; IDTOligo Analyzer at 207.32.43.70/biotools/ DNA, and linear DNA or RNA. In one embodiment, DL oligocalc/oligocalc.asp; Mfold at bioinfo.rpi.edu/applica NAMs are comprised entirely of nucleic acids that are tions/mfold/; and WWTACGV2.38 at koubai.virus.kyotou. Y-shape. In a further embodiment, DL-NAMs are comprised ac.jp/tacg2/tacg2.form.html. of Y-shape and X-shape nucleic acids, in a ratio that is prese lected as desired. 0104 One of the easiest and informative ways to charac 0102. In one embodiment, DL-NAMs are assembled by terize the seuqences and the formation of dendrimer-like ligation of Y-DNA molecules, whose sequences are specifi DNA is the conventional agarose gel electrophoresis. It is also cally designed so that ligations between Yi and Y-DNA could possible to visualize the structure using high resolution only occur when izi, where i and j refer to the generation microscopytechniques such as TEM or AFM, where the final number n, for example, G1, G2,etc. The cohesive end of each structure is large enough to be resolved by Such instruments. oligonucleotide is non-palindromic, thus no self-ligations 0105. In one example, 4 consecutive nucleotides were occurred. In addition, the ligation can only occur in one used as a unit in the checking process. For example, Target direction, that is, Y->Y->Y->Y and so on. Furthermore, sequence: AGCTGAT when Yo is ligated to Y with a 1:3 molar stoichiometry, one 0106 Check 1: AGCT. Since no other AGCT sequence Yo was linked with three Y, forming the first-generation appears in that sequence, the first sequence symmetry check DL-DNA. G1 is then ligated to six Y (one Y for each of the passes.

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T-Shape TABLE 4B- continued 0118. In yet another aspect, the nucleic acids forming a Sequence Table matrix are T-shape nucleic acids (FIG. 10). In one embodi ment, the T-shape nucleic acids are DNA. In yet another SEQ ID NO Sequence embodiment, the matrix comprises T-shape DNA and/or 38 5 - GTCA-3' RNA, or analogs/derivatives thereof. In addition, a matrix can 39 5 - CGAT-3' be comprised of T-shape and one or more different shapes of nucleic acids, including X-, Y-, dumbbell- or dendrimer 4 O 5-ATCG-3 shape nucleic acids, as well as a a combination thereof. 41 5 - GCAT-3' 0119. In one embodiment, the T-shape nucleic acids have a tensile strength selected from 50, 51, 52,53,54, 55, 56, 57. 42 S " — ATGC - 3 " 58, 59, 60, 61, 62, 63, 64, or 65%. In addition, the T-shape 43 5 " - TTGC-3 " nucleic acids can have a degree of Swelling selected from 100, 105, 110, 115, 120, 125, 130, or 135%. For the T-shaped 44 S " — GCAA- 3 " nucleic acid molecule, the second region (region 2) of each 45 5 - GGAT-3' polynucleotide is complementary to the fourth region (region 4) of one of the other two polynucleotides. The fourth region 46 5-TGGATCCGCATGA-3 of each polynucleotide is complementary to the second region of the other of the other two polynucleotides of 47 5 * - CATTCGCCGTAAG-3 * T-shaped nucleic acid molecule. The third region is either 48 S " - CTTACGGCGAATG- 3 " absent or is a linker to permit formation of the T-shaped configuration. For example, with reference to the sequences 49 5'-ACCGAATCAGCCT-3 in Tables 4 and 5A: region 2 of SEQID NO: 36 is comple SO 5'-AGGCTGATTCGGT-3 mentary to region 4 of SEQID NO:34, region 4 of SEQID NO:36 is complementary to region 2 of SEQID NO:35, and 51 5-TCATGCGGATCCA-3 region 2 of SEQID NO. 34 is complementary to region 4 of 52 TTGCTGGATCCGCATGACATTCGCCGTAAG-3 * SEQID NO:35. 53 CGTTTGGATCCGCATGACATTCGCCGTAAG-3 * I0120 T-shaped nucleic acid molecules can be synthesized by mixing equal amounts of three oligonucleotide strands. 54 ATGCTGGATCCGCATCGACATTCGCCGTTAAG — 3 " The nomenclature is as follows: To To, and To, are the 55 TGGATCCGCATGACATTCGCCGTAAG – 3 * three corresponding single oligonucleotide chains that forma To-nucleic acid molecule (To). Similarly, T.T., and Tare 56 GCATCTTACGGCGAATGACCGAATCAGCCT-3 the three corresponding single oligonucleotide chains that form a T-nucleic acid molecule (T); and T. T., and T. 57 GCAACTTACGGCGAATGACCGAATCAGCCT-3 are the three corresponding single oligonucleotide chains that 58 CTTACGGCGAATGACCCGAATCACGCCT - 3 " form a T-shaped nucleic acid molecule (T). The reactions can be the following: To-To-To->To, T+T+T sT, 59 (GCATAGCCT(GATT COGGT'''T'CATGCGGATCCA. - 3 ' and T+T+T->T, etc. (see FIGS. 10 and 26). 6 O TTGCACGGCTGATTCGGTTC ATGCGGATCCA — 3 " 0.121. In various embodiments, selection of X-, Y- or 61 AACGAGGCTGATTCGGTTCATGCGGATCCA- 3 " T-DNA can be utilized to design hydrogels of differing exter nal morphologies and internal structure. (e.g., FIGS. 1, 2 and 62 ACGGCTGATTCGGTTCATGCGGATCCA- 3 " 5). For example, in a dry state surface morphology revealed a tangled pattern for X-DNA gel (FIG. 5A), a fibrous form for Y-DNA gel, and a scale shape for T-DNA gel. (FIG. 5). 0115. In another aspect, the matrix is comprised of Furthermore, X-DNA gels can exhibit two flat DNA gel strips Y-shape and linear nucleic acids. In one embodiment, the tanbled into a knot to form a large sheet with many wrinkles Y-shape and linear nucleic acid is DNA. In another embodi on the surface.Y-DNA gel (FIG.5B) exhibits fibrous a fibrous ment, the Y-shape nucleic acid is DNA, while the linear form spreading out from many branches. T-DNA gel (FIG. nucleic acid is DNA, RNA, TNA or PNA. In one embodi 5C) exhibits puckers on a sheet. In a swollen state, the surface ment, the Y-shaped nucleic acids are DNA. In another, morphology of the gels exhibited a large number of various embodiment, the Y-shaped nucleic acids are RNA. sized pores and channels (FIG. 5D), with obvious fibers of I0116). In certain aspects of the invention, the Y-DNA ter fractal-shapes on the periphery (FIG. 5E) and perpendicu minal ends are designed with Sticky ends as described above larly erected, scale like structures (FIG. 5F) for X-, Y- and that are capable of undergoing an enzymatic reaction. In one T-DNAS. embodiment, the enzymatic reaction is a ligation reaction I0122. In yet other embodiments, gels can be comprised of with a DNA ligase. In yet a further embodiment, the DNA one or more differently shaped nucleic acids, including X-, ligase is a T4 DNA ligase. Y-, T., dumbbell- or dendrimer-shaped DNA (e.g., Y- and 0117. In one embodiment, Y-shape nucleic acid building X-DNA, or Y- and T-DNA or X- and T-DNA). In yet further blocks are joined end-to-end to produce a dumbell shaped embodiments, applicable to any matrix disclosed herein, gels building block or dendrimer like nucleic acid. (e.g., FIGS. 9 can be comprised of nucleic acids that include DNA, RNA, and 30B) PNA, TNA, or a combination thereof. US 2010/013.6614 A1 Jun. 3, 2010 13

invention, dendrimer like nucleic acid structures are TABLE 5 assembled to provide a biomaterial compound. I0128. In other aspects of the invention branched or DL Example of Oligonucleotides used to construct NAMs are utilized to form dendrimer structures. Synthesiz X-, Y- and T-nucleic acid building blocks. ing monodisperse polymers demands a high level of synthetic SEQ ... ID . control which is achieved through stepwise reactions, build Strand Segment Segment 2 NO. ing the dendrimer up one monomer layer, or 'generation, at Xo1 5 " – p – ACGT CGA CCG ATG AAT AGC GGT 63 a time. Each dendrimer consists of a multifunctional core CºAG ATC CGT ACC TAC TGG-3 ! molecule with a dendritic wedge attached to each functional site. The core molecule is referred to as “generation 0. Each Xo2 5 -p-ACGT CGA GTA GGT ACG GAT CTG 64 Successive repeat unit along all branches forms the next gen CGT ATT GCG AAC GAC TGG-3 eration, “generation 1. “generation 2. and so on until the Xo3 5 -p-ACGT CGA GTC GTT CGC AAT ACG 65 terminating generation (e.g., FIG. 4A and FIG. 7). Such a GCT GTA CGT ATG GTC TCG-3' level of control is achieved through controlled assembly XO4 5' -p-ACGT CGA GAC CAT ACG TAC AGC 66 through the sticky ends of the multimer building blocks uti ACC GCT ATT CAT CGG TCG-3' lized to assemble a DL-NAM. I0129. There are two defined methods of dendrimer syn Y, 5 " – p – ACGT CGA CCG ATG AAT AGC GGT 67 thesis, divergent and convergent. In the divergent method the CºAG ATC CGT ACC TAC TCG – 3 º molecule is assembled from the core to the periphery; while in ??, 5 -p-ACGT CGA GTC GTT CGC AAT ACG 68 the convergent method, the dendrimer is synthesized begin ACC GCT ATT CAT CGG TCG-3' ning from the outside and terminating at the core. In either method the synthesis requires a stepwise process, attaching ?? 5 -p-ACGT CGA GTA GGT ACG GAT CTG 69 one generation to the last, purifying, and then changing func CGT ATT GCG AAC GAC TCG-3 tional groups for the next stage of reaction. For example, in T 5 -p-ACGT CGA CAG CTG ACT AGA GTO 70 FIG. 7, the shaded inner core represents one step, followed by ACG ACC TGT ACC TAC TCG-3' the unshaded “Y” molecules as an additional and subsequent T. 5'-p-ACGT CGA GTG GTT CTC AAG ACG 71. step, and finally the stipeled “Y” molecules as a further addi TAG CTA GGA CTC TAG TCA tional and Subsequent step. This functional group transforma GCT GTC G — 3 " tion is necessary to prevent unbridled polymerization. Such polymerization can lead to a highly branched molecule which ?. 5'-p-ACGT CGA GTA GGT ACA GGT CGT 72 is not monodisperse—otherwise known as a hyperbranched CGT CTT GAG AAC GAC TCG-3 polymer. 0.130. In the divergent method, the surface groups initially 0123 Note that p represents the phosphorylation on the 5' are unreactive or protected species which are converted to end of the oligonucleotide. reactive species for the next stage of the reaction. In the 0124. To confirm the formation of these branched DNA convergent approach the opposite holds, as the reactive spe building blocks, a gel electrophoretic migration-shift assay cies must be on the focal point of the dendritic wedge. (GEMSA) coupled with a DNA-specific fluorescent dye I0131 Due to steric effects, continuing to react dendrimer (SYBRI) was employed. In general, lower salt concentra repeat units leads to a sphere shaped or globular molecule tions can be used for more specific base-pairing, while higher until steric overcrowding prevents complete reaction at a salt concentrations favor Strong electro-static intereactions. specific generation and destroys the molecule's monodisper Ionic influences on DNA are familiar to one of ordinary skill sity. The number of possible generations can be increased by in the art. (See, e.g., Macromolecules, 1997, 30: 5763; J. using longer spacing units in the branches of the core mol Phys. Chem. 2006: 110: 2918-2926: Biophys.J. 1996; 70: ecule. The monodispersity and spherical steric expansion of 2838-46. dendrimers leads to a variety of interesting properties. The 0.125 Similar experiments as above were also performed steric limitation of dendritic wedge length leads to Small with X-Y- and T-DNA, which led to controlled-assembled of molecular sizes, but the density of the globular shape leads to three dimensional structures. For ligation, manufacturer's fairly high molecular weights. The spherical shape also pro protocols were followed. Mg++ was added for ATP. Hydrogel vides an interesting study in molecular topology. Dendrimers gelation correlated with ligase activity. For example, by using have two major chemical environments, the Surface chemis twice the amount of ligase (e.g., 60 Units), the DNA hydrogel try due to the functional groups on the termination generation, was completely formed within 30 minutes. A typical example which is the surface of the dendritic sphere, and the sphere's of ligase reaction utilized Ligase 10x buffer which has a interior which is largely shielded from exterior environments composition of 300 mM Tris-HCl (pH 7.8), 100 mMMgCl2, due to the spherical shape of the dendrimer structure. The 100 mM DTT and 10 mM ATP T4 DNA ligase is supplied existence of two distinct chemical environments in Such a with 10 mMTris-HC1 (pH 74), 50 mMKCl, 1 mM DTT 0.1 molecule implies many possibilities for dendrimer applica mM EDTAand 50% glycerol. tions. 0126 Dendrimer Structures 0.132. As such, hydrophobic/hydrophilic and polar/nonpo 0127. As almost all nucleic acid molecules are either lin lar interactions can be varied in the two environments. The ear or circular, to rationally construct nucleic acid biomate existence of voids in the dendrimer interior furthers the pos rials, additional shapes of nucleic acids as basic building sibilities of these two heterogeneous environments playing an blocks must be first constructed. In addition, these nucleic important role in dendrimer chemistry. Therefore, in a further acid building blocks must be readily incorporated into larger embodiment dendrimer structures can accept molecules in structures in a controlled manner. Thus, in one aspect of the the Void spaces in addition to or alternative to the linkage to

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0145 Therefore, in some embodiments, DL-NAMs carry 0151. In some embodiments, DNA condensing peptides an anticancer drug and a targeting moiety to a cancer cell. can be linked to DL-NAMs. For example, with advancement whereby targeted drug delivery is founded on the principle of genetic engineering and protein chemistry, many peptides that if a receptor is expressed specifically or in excess on the can be designed de novo and synthesized accordingly. The Surface of a cancer cell, the dendrimer carrying a drug and a condensing peptide (Y-WKC) has been successfully utilized ligand for the receptor travels through the circulation, binds for DNA delivery as a synthetic, DNA condensation peptide. specifically to the cancerous cells, and delivers the drug to 0152. In another embodiment, the Y-shape, T-shape, induce programmed cell death. Furthermore, owing to the X-shape or dumbbell shape nucleic acids are linked to one or multivalent character of the DL-NAMs of the invention con more biologically active agents, including the preceding pep jugation of several molecules of a targeting agent onto the tides, one or more selection markers, one or more detectable dendrimer will result in an increase in the dendrimer's avidity labels, one or more drugs, Small compounds, or nucleic acid for binding to the targeted cells through multivalent interac sequences or one or more copolymer compounds. tion because of the binding of multiple targeting molecules to 0153. In certain embodiments, the dendrimer structures their receptor. While not limited to any particular cancer are linked or cross-linked to additional compounds selected expressed marker, in some embodiments, DL-NAMs com from a group consisting of an adenovirus core peptide, a prise a targeting moiety that is Folic acid receptor (FAR), synthetic peptide, an influenza virus HA2 peptide, a simian which is overexpressed on the Surface of a variety of malig immunodeficiency virus gp32 peptide, an SV40 T-Ag pep nancies, such as cancer of the head and neck and of the ovary. tide, a VP22 peptide, a Tat peptide, and a Rev peptide. Such 0146 In one embodiment the Y-shape, X-shape, T-shape additional compounds are selected from a group consisting of or dumbbell-shape arms are attached to a peptide moiety DNA condensing peptide, DNA protection peptide, endoso comprising an adenovirus core peptide, a synthetic peptide, mal targeting peptide, membrane fusion peptide, nuclear an influenza virus HA2 peptide, a simian immunodeficiency localization signaling peptide, a protein transduction domain virus gp32 peptide, an SV40 T-Ag peptide, a VP22 peptide, a peptide or a combination thereof (FIGS. 18-19). Tat peptide, a Rev peptide, DNA condensing peptide, DNA 0154. In one embodiment, the dendrimer structures are protection peptide, endosomal targeting peptide, membrane utilized in a method of delivering a biologically active agent fusion peptide, nuclear localization signaling peptide, a pro to a cell, or to a subject. In another embodiment, the den tein transduction domain peptide or any combination thereof. drimer structure comprises a linkage to a signal or targeting 0147 Such peptides can be selected based on their prop peptide as described herein above, as well as a bioactive agent erties related including but not limited to properties to over having therapeutic properties (e.g., drug, siRNA, nucleic acid come various cellular barriers, peptides from viruses chosen encoding a therapeutic protein). Additional targeting and from disparate viruses to preclude capsidassembly, providing delivery moieties are known in the art, such as disclosed in an extra amino acid Cys at C-terminal end to facilitate con U.S. Pat. No. 7,122,525; 7,122,172, 7,097.856, 699176, jugation or pre-determining ratios of peptide to DL-NAM to 6992.169, 6977075 or 6939528. provide DL-NAMs with specific peptides linked thereonto. 0155 Labels and Selection Markers 0148. Nonlimiting examples of peptides include, SV40 0156. In yet another embodiment, the dendrimer com NLS peptide or semian virus 40 large tumor antigen prises atargeting peptide, a biologically active agent, a Selec (FKKKRKVEDPYC; SEQ ID NO: 73), which is a nuclear tion marker and a detectable label. Selection markers include localization peptide (NLS) that can translocate other mol antibiotics which are known in the art for both eukaryotic and ecules from cytosol to the nucleus through the nuclear mem prokaryotic cells, or disclosed herein. Infra. Therefore, as brane. noted above, a dendrimer can provide a multivalent strucutre 0149. In some embodiments the delivery peptide is HIV comprised of several distinct molecules that are bound to one Tat. The trans-activating transcriptional activator (Tat) which or more arms of a one or more multimer nucleic acid mol is an 86 amino acid protein from HIV-1. The effective part of ecules of which a dendrimer is composed. (e.g., FIGS. Tat for translocation can be as short as 13 amino acids 18-20). (TAT48-60: GRKKRRQRRRPPQ: SEQ ID NO: 74). Tat can 0157 Specific examples of detectable molecules include offer efficient intracellular delivery of both macromolecules radioactive isotopes such as P’ or H. fluorophores such as and small particles. HIV Tat is ideal to overcome the plasma fluorescein isothiocyanate (FITC) FIG. 20, TRITC, uptake barrier. rhodamine, tetramethylrhodamine, R-phycoerythrin, Cy-3, 0150. In some embodiments the delivery peptide is Adno Cy-5, Cy-7, Texas Red, Phar-Red, allophycocyanin (APC), mu peptide. Adenovirar core peptide mu (MRRAHIHIR epitope tags Such as the FLAG or HA epitope, and enzyme RRRASHRRMRGG: SEQID NO:75) functions as a nucleic tags such as alkaline phosphatase, horseradish peroxidase, acid condensing peptide and can be used to condense DNA If-galactosidase, and hapten conjugates such as digoxigenin for gene delivery due to its highly cationic properties. An or dinitrophenyl, etc. Other detectable markers include important aspect of the invention is not a particular targeting chemiluminescent and chromogenic molecules, optical or peptide, but the multivalency of DL-NAMs. For example, an electron density markers, etc. The probes can also be labeled artisan will realize that receptors represent a simple alterna with semiconductor nanocrystals such as quantum dots (i.e., tive to the use of antibodies as targeting ligands for cell Qdots), described in U.S. Pat. No. 6,207,392. Qdots are com specific gene delivery, although antibodies are readily adapt mercially available from Quantum Dot Corporation. able to DL-NAMs of the invention. Additional cellular tar 0158. Additional examples of reagents which are useful geting peptides are known to one of skill in the art, Such as for detection include, but are not limited to, radiolabeled disclosed in U.S. Pat. Nos. 6.649,407; 6,576,456; 6,548,634; probes, fluorophore-labeled probes, quantum dot-labeled See also, Aronsohn A II and Hughes J A. Nuclear localization probes, chromophore-labeled probes, enzyme-labeled signal peptides enhance cationic liposome-mediated gene probes, affinity ligand-labeled probes, electromagnetic spin therapy. J Drug Target. 1998; 5:163-169. labeled probes, heavy atom labeled probes, probes labeled US 2010/013.6614 A1 Jun. 3, 2010

with nanoparticle light scattering labels or other nanopar 0163 Thus, in another aspect of the invention, the thera ticles or spherical shells, and probes labeled with any other peutic capable agent is a bioactive protein or peptide. signal generating label known to those of skill in the art. Examples of such bioactive protein or peptides include a cell Non-limiting examples of label moieties useful for detection modulating peptide, a chemotactic peptide, an anticoagulant in the invention include, without limitation, Suitable enzymes peptide, an antithrombotic peptide, an anti-tumor peptide, an Such as horseradish peroxidase, alkaline phosphatase, B-ga anti-infectious peptide, a growth potentiating peptide, and an lactosidase, or acetylcholinesterase; members of a binding anti-inflammatory peptide. Examples of proteins include pair that are capable of forming complexes such as Streptavi antibodies, enzymes, steroids, growth hormone and growth din/biotin, avidin/biotin or an antigen/antibody complex hormone-releasing hormone, gonadotropin-releasing hor including, for example, rabbit IgG and anti-rabbit IgG; fluo mone, and its agonist and antagonist analogues, Somatostatin and its analogues, gonadotropins such as luteinizing hormone rophores such as umbelliferone, fluorescein, fluorescein and follicle-stimulating hormone, peptide T, thyrocalcitonin, isothiocyanate, rhodamine, tetramethyl rhodamine, eosin, parathyroid hormone, glucagon, Vasopressin, oxytocin, green fluorescent protein, erythrosin, coumarin, methyl cou angiotensin I and II, bradykinin, kallidin, adrenocorticotropic marin, pyrene, malachite green, stilbene, lucifer yellow, Cas hormone, thyroid stimulating hormone, insulin, glucagon and cade BlueTM, Texas Red, dichlorotriazinylamine fluorescein, the numerous analogues and congeners of the foregoing mol dansyl chloride, phycoerythrin, fluorescent lanthanide com ecules. The therapeutic agents may be selected from insulin, plexes such as those including Europium and Terbium, Cy3, antigens selected from the group consisting of MMR Cy5, molecular beacons and fluorescent derivatives thereof, (mumps, measles and rubella) vaccine, typhoid vaccine, as well as others known in the art as described, for example, hepatitis A vaccine, hepatitis B vaccine, herpes simplex virus, in Principles of Fluorescence Spectroscopy, Joseph R. bacterial toxoids, cholera toxin B-subunit, influenza vaccine Lakowicz (Editor), Plenum Pub Corp, 2nd edition (July 1999) Virus, bordetela pertussis virus, Vaccinia virus, adenovirus, and the 6' Edition of the Molecular Probes Handbook by canary pox, polio vaccine virus, plasmodium falciparum, Richard P. Hoagland; aluminescent material Such as luminol; bacillus calmette geurin (BCG), klebsiella pneumoniae, HIV light scattering or plasmon resonant materials such as gold or envelop glycoproteins and cytokins and other agents selected silver particles or quantum dots; or radioactive material from the group consisting of bovine somatropine (sometimes include 14C, 2I, 12*I, 125I, II, Tc99m, SS or H. referred to as BST), estrogens, androgens, insulin growth 0159. Examples of labels include, but are not limited to, factors (sometimes referred to as IGF), interleukin I, interleu chromophores, fluorescent moieties, enzymes, antigens, kin II and cytokins. Three such cytokins are interferon-a, heavy metal, magnetic probes, dyes, phosphorescent groups, interferon-band tuftsin. radioactive materials, chemiluminescent moieties, Scattering 0164. In one embodiment a cell modulating peptide is or fluorescent nanoparticles, Raman signal generating moi selected from the group consisting of an anti-integrin anti eties, and electrochemical detection moieties. Genotyping body fragment, a cadherin binding peptide, a bone morpho using a microarray can be performed using any of a variety of genic protein fragment, and an integrin binding peptide. Pref methods, means and variations thereof for carrying out array erably the cell modulating peptide is a integrin binding genotyping analysis. peptide which is selected from the group consisting of 0160. Furthermore, backbone labels are nucleic acid RGDC, RGEC, RGDT, DGEA, DGEAGC, EPRGDNYR. stains that bind nucleic acid molecules in a sequence inde RGDS, EILDV. REDV, YIGSR, SIKVAV, RGD, RGDV. pendent manner. Examples include intercalating dyes such as HRNRKGV, KKGHV. XPQPNPSPASPVVVGG phenanthridines and acridines (e.g., ethidium bromide, pro GASLPEFXY, and ASPVVVGGGASLPEFX. The peptides pidium iodide, hexidium iodide, dihydroethidium, ethidium also may be any functionally active fragment of the proteins homodimer-1 and -2, ethidium monoazide, and ACMA): disclosed herein as being bioactive molecules useful accord Some minor grove binders such as indoles and imidazoles ing to the invention. In another embodiment the chemotactic (e.g., Hoechst 33258, Hoechst 33342, Hoechst 34580 and peptide is selected from the group consisting of functionally DAPI); and miscellaneous nucleic acid stains such as acridine active fragments of collagen, fibronectin, laminin, and pro orange (also capable of intercalating), 7-AAD, actinomycin teoglycan. In yet another embodiment the anti-tumor peptide D. LDS751, and hydroxy stilbamidine. All of the aforemen is selected from the group consisting of functionally active tioned nucleic acid stains are commercially available from fragments of protein anti-tumor agents. The anti-infectious suppliers such as Molecular Probes, Inc. Still other examples peptide is selected from the group consisting of functionally of nucleic acid stains include the following dyes from active fragments of the protein anti-infectious agents accord Molecular Probes: cyanine dyes such as SYTOX Blue, ing to another embodiment. In another embodiment the SYTOX Green, SYTOX Orange, POPO-1, POPO-3, YOYO growth potentiating peptide is selected from the group con 1,YOYO-3, TOTO-1, TOTO-3, JOJO-1, LOLO-1, BOBO-1, sisting of functionally active fragments of PDGF, EGF, FGF, BOBO-3, PO-PRO-1, PO-PRO-3, BO-PRO-1, BO-PRO-3, TGF, NGF, CNTF, GDNF, and type I collagen related pep TO-PRO-1, TO-PRO-3, TO-PRO-5, JO-PRO-1, LO-PRO-1, tides. According to another embodiment the anti-inflamma YO-PRO-1, YO-PRO-3, PicoGreen, OliGreen, RiboGreen, tory peptide is selected from the group consisting of function SYBR Gold, SYBR Green I, SYBR Green II, SYBR DX, ally active fragments of anti-inflammatory agents. SYTO-40, -41, -42, -43, -44, -45 (blue), SYTO-13, -16, -24, 0.165. Other bioactive peptides useful according to the -21, -23, -12, -11, -20, -22, -15, -14, -25 (green), SYTO-81, invention may be identified through the use of synthetic pep -80, -82, -83, -84, -85 (orange), SYTO-64, -17, -59, -61, -62, tide combinatorial libraries such as those disclosed in Hough -60, -63 (red). ton et al., Biotechniques, 13(3):412-421 (1992) and Hough 0161 Therapeutic Polypeptides ton et al., Nature, 354:84-86 (1991) or using phage display 0162. As will not be apparent, DL-NAMs can be linked procedures such as those described in Hart, et al., J. Biol. with any therapeutic compound(s) including polypeptides. Chem. 269:12468 (1994). Hart et al. report a filamentous US 2010/013.6614 A1 Jun. 3, 2010 phage display library for identifying novel peptide ligands for for protein-protein and protein-oligonucleotide conjugation mammalian cell receptors. In general, phage display libraries between an NH group and a SH group. SMCC has n using, e.g., M13 or fa phage, are prepared using conventional NHS-ester and a maleimide group, which result in primary procedures such as those described in the foregoing refer amine and sulfhydryl reactivity. The cyclohexane bridge ence. The libraries display inserts containing from 4 to 80 makes the maleimide group extra stable (See, Example amino acid residues. The inserts optionally represent a com pletely degenerate or a biased array of peptides. Ligands that below). bind selectively to a specific molecule Such as a cell Surface 0171 In some embodiments, DL-NAMs can comprise receptor are obtained by selecting those phages which nucleic acids encoding therapeutic products. Nonlimiting express on their Surface a ligand that binds to the specific examples of Such nucleic acids include ones encoding inter molecule. Ligands that possess a desired biological activity feron, interleukin, erythropoietin, granulocyte-colony stimu can be screened in known biological activity assays and lating factor (GCSF), stem cell factor (SCl:), leptin (OB pro selected on that basis. These phages then are Subjected to tein), interferon (alpha, beta, gamma), ciprofloxacin, several cycles of reselection to identify the peptide-express amoxycillin, lactobacillus, cefotaxime, levofloxacin, ceflip ing phages that have the most useful characteristics. Typi ime, mebendazole, ampicilin, lactobacillus, cloxacilin, nor cally, phages that exhibit the binding characteristics (e.g., floxacin, timidazole, cdpodoxime, proxctil, azithromycin, highest binding affinity or cell stimulatory activity) are fur gatifloxacin, roxithromycin, cephalosporin, anti-thrombo ther characterized by nucleic acid analysis to identify the genics, aspirin, ticlopidine, Sulfinpyrazone, heparin, war particular amino acid sequences of the peptides expressed on farin, growth factors, differentiation factors, hepatocyte the phage surface and the optimum length of the expressed stimulating factor, plasmacytoma growth factor, brain peptide to achieve optimum biological activity. Alternatively, derived neurotrophic factor (BDNF), glial derived neu such peptides can be selected from combinatorial libraries of rotrophic factor (GDNF), neurotrophic factor 3 (NT3), fibro peptides containing one or more amino acids. Such libraries blast growth factof FGF), transforming growth factor (TGF). can further be synthesized which contain non-peptide Syn platelet transforming growth factor, milk growth factor, thetic moieties which are less Subject to enzymatic degrada endothelial growth factors (EGF), endothelial cell-derived tion compared to their naturally-occurring counterparts. U.S. growth factors (ECDGF), alpha-endothelial growth factors, beta-endothelial growth factor, neurotrophic growth factor, Pat. No. 5,591,646 discloses methods and apparatuses for nerve growth factor (NGF), vascular endothelial growth fac biomolecular libraries which are useful for screening and tor (VEGF), 4-1 BB receptor (4-1BBR), TRAIL (TNF-re identifying bioactive peptides. Methods for screening pep lated apoptosis inducing ligand), artemin (GFRalpha3-RET tides libraries are also disclosed in U.S. Pat. No. 5,565,325. ligand), BCA-1 (B cell-attracting chemokinel), B lympho (0166 Nucleic Acid Delivery cyte chemoattractant (BLC), B cell maturation protein 0167. In yet another aspect of the invention, the dendrimer (BCMA), brain-derived neurotrophic factor (BDNF), bone (DL-NAM) is linked to therapeutic nucleic acids, including growth factor such as osteoprotegerin (OPG), bone-derived linear or branched nucleic acids, genes/antigenes, nucleic growth factor, megakaryocyte derived growth factor acid vectors (e.g., plasmid or viral vectors or linear nucleic (MGDF), keratinocyte growth factor (KGF), thrombopoietin, acid sequences), all of which are delivered into a cell or platelet-derived growth factor (PGDF), megakaryocyte subject utilizing DL-NAMs which can be “loaded with one derived growth factor (MGDF), keratinocyte growth factor or more therapeutic "payloads”. (KGF), platelet-derived growth factor (PGDF), bone mor 0168 Thus in some embodiments, the dendrimer struc phogenetic protein 2 (BMP2), BRAK, C-10, Cardiotrophin 1 tures are used in method of effecting transfection or genetic (CT1), CCR8, anti-inflammatory: paracetamol, salsalate, modification of a cell. One central aspect of the dendrimer diflunisal mefenamic acid, diclofenac, piroxicam, ketopro structures are anisotropic and multivalent. Such DL-NAMs fen, dipyrone, acetylsalicylic acid, antimicrobials amoxicil can be administered to a cell or Subject using methods known lin, ampicillin, cephalosporins, erythromycin, tetracyclines, in the art for delivery of nucleic acids (e.g., plasmids or viral penicillins, trimethprim-sulfamethoxazole, quiniolones, vectors), such as disclosed in U.S. Pat. No. 6,946,448; 6,893, amoxicillin, clavulanatfazithromycin, clarithromycin, anti 664; 6,821,955; 6,689,757; 6,562,801; 6,951,755; 6,841,540: cancer drugs aliteretinoin, altertamine, anastroZole, azathio 6,818,213 or 6,649,407. prine, bicalutamide, busulfan, capecitabine, carboplatin, cis 0169. In one embodiment, DL-NAMs are comprised of platin, cyclophosphamide, cytarabine, doxorubicin, one or more biologically active agents to be delivered alone or epirubicin, etoposide, exemestane, Vincristine, vinorelbine, in combination with another compound to a cell or Subject. In hormones, thyroid stimulating hormone (TSH), sex hormone order to use DL-NAMs as scaffolding for multi-functional binding globulin (SHBG), prolactin, luteotropic hormone modules (e.g., bioactive agents such as peptides), the nucleic (LTH), lactogenic hormone, parathyroid hormone (PTH), acids comprising DL-NAMs can be functionalized with the melanin concentrating hormone (MCH), luteinizing hormone desired bioactive agent. Nucleic acid molecules have been (LHb), growth hormone (HGH), folliclestimulating hormone conjugated with many chemical moieties, thus effectively (FSHb), haloperidol, indomethacin, doxorubicin, epirubicin, linking diverse chemical functionalities (Zhu et al., J. An amphotericin B, Taxol, cyclophosphamide, cisplatin, meth Chem Soc 125, 10178 (2003) otrexate, pyrene, amphotericin B, anti-dyskinesia agents, 0170 In one example, DNA is amine-modified at the com Alzheimer vaccine, antiparkinson agents, ions, edetic acid, mercial synthesis stage. Peptides are synthesized with an nutrients, glucocorticoids, heparin, anticoagulation agents, extra Cys at their C-terminal. Furthemore, many homo- and anti-virus agents, anti-HIV agents, polyamine, histamine and hetero-bi-functional crosslinkers can be used for protein derivatives thereof, cystineamine and derivatives thereof, nucleic acid conjugation. Examples of Some common cross diphenhydramine and derivatives, orphenadrine and deriva linkers include succinimdyl 4-(N-maleimidomethyl)cyclo tives, muscarinicantagonist, phenoxybenzamine and deriva hexane-1-carboxylate (SMCC), which is widely employed tives thereof, protein A, Streptavidin, amino acid, beta-galac US 2010/013.6614 A1 Jun. 3, 2010

tosidase, methylene blue, protein kinases, beta-amyloid, PNAS USA 77:4216-4220 (1980), which are hereby incor lipopolysaccharides, eukaryotic initiation factor-4G, tumor porated by reference. The vector used in one or methods necrosis factor (TNF), tumor necrosis factor-binding protein disclosed herein may be a viral vector, preferably a retroviral (TNF-bp), interleukin-1 (to 18) receptor antagonist (IL-Ira), vector. Replication deficient adenoviruses are preferred. For granulocyte macrophage colony stimulating factor (GM example, a “single gene vector” in which the structural genes CSF), novel erythropoiesis stimulating protein (NESP), of a retrovirus are replaced by a single gene of interest, under thrombopoietin, tissue plasminogen activator (TPA), uroki the control of the viral regulatory sequences contained in the nase, streptokinase, kallikrein, insulin, steroid, acetylsali long terminal repeat, may be used, e.g. Moloney murine cylic acid, acetaminophen, analgesic, anti-tumor preparation, leukemia virus (MoMulV), the Harvey murine sarcoma virus anti-cancer preparation, anti-proliferative preparation or pro (HaMuSV), murine mammary tumor virus (MuMTV) and apoptotic preparation. the murine myeloproliferative sarcoma virus (MuMPSV). 0172. In some aspects DL-NAMs can be linked to a plas and avian retroviruses such as reticuloendotheliosis virus mid or viral vector which itself encodes a therapeutic gene. (Rev) and Rous SarcomaVirus (RSV), as described by Eglitis Examples of such plasmid or viral vectors include adenoviral and Andersen, BioTechniques 6(7):608-614 (1988), which is vectors, adenoviral associated vectors, retroviral vectors, hereby incorporated by reference. and/or eukaryotic cell plasmid vectors FIG. 19, which can 0.178 Recombinant retroviral vectors into which multiple further encode any therapeutic gene of interest. In various genes may be introduced may also be used with the matrixes embodiments, a DL-NAM can be linked to one or more or methods of the invention. As described by Eglitis and desired nucleic acid for delivery to a target cell. Examples of Andersen; above, vectors with internal promoters containing Such nucleic acids include genes and antigenes, siRNA, a cDNA under the regulation of an independent promoter, e.g. RNAi, nucleic acids. SAX vector derived from N2 vector with a selectable marker 0173. In various embodiments, DL-NAMs are comprised (noe. sup.R) into which the cDNA for human adenosine of one or more targeting moieties, in addition to a therapeutic deaminase (haDA) has been inserted with its own regulatory payload (e.g., genes or antigenes). Targeting moieties are sequences, the early promoter from SV40 virus (SV40) may disclosed herein and known in the art, and result in enhanced be designed and used in accordance with methods disclosed cellular uptake and release of a therapeutic payload. Further herein oras known in the art. more, based on the inherent properties of DL-NAMs, there is 0179. In some aspects of the invention, the vectors com reduced toxicity and adverse effects associated with viral prising recombinant nucleic acid molecules are first intro delivery vectors. Moreover, DL-NAMs provide modular/ duced (e.g., transfected) into cells, which cells are deposited multivalent functionality, where for example, targeting and in the matrixes of the invention. For example, the vectors therapeutic (as well as detectable and selectable) compounds comprising the recombinant nucleic acid molecule are incor can be linked to a particular DL-NAM (FIGS. 18-20). porated, i.e. infected, into the BM-MNCs by plating ~5e5 0.174 Delivery can be to eukaryotic or prokaryotic cells. BM-MNCs over vector-producing cells for 18-24 hours, as Furthermore, delivery can be to mammalian cells or animals. described by Eglitis and Andersen BioTechniques 6(7):608– Furthermore, delivery can be to species of animals including 614 (1988), which is hereby incorporated by reference, and but not limited to simian, human, murine, bovine, equine, Subsequently said cells are deposited into the reservoir por bird, reptile or insects. tion of the device. 0.175. In yet other aspects of the invention, one or more 0180. In mammalian host cells, a number of viral-based vectors each encoding a different therapeutic capable agent expression systems can be utilized. In cases where an aden delivered to cells or tissue via DL-NAMs of the invention. ovirus is used as an expression vector, the nucleotide Such delivery can be of plasmid vectors that afford endog sequence of interest (e.g., encoding a therapeutic capable enous control via promoters which are sensitive to a physi agent) can be ligated to an adenovirus transcription or trans ological signal Such as hypoxia or glucose elevation. Further lation control complex, e.g., the late promoter and tripartite more, such plasmid vectors can afford exogenous control leader sequence. This chimeric gene can then be inserted in systems for gene expression controlled from without the cell, the adenovirus genome by in vitro or in Vivo recombination. for example, by administering a small molecule drug. Insertion in a non-essential region of the viral genome (e.g., Examples include tetracycline, doxycycline, ecdysone and its region E1 or E3) will result in a recombinant virus that is analogs, RU486, chemical dimerizers such as rapamycin and viable and capable of expressing the AQP1 gene product in its analogs, etc. infected hosts. (See e.g., Logan & Shenk, Proc. Natl. Acad. 0176). In an alternative aspect of the invention, DL-NAMs Sci. USA 81:3655-3659 (1984)). can deliver one or more drug, Such as those described herein, 0181. In one embodiment, DL-NAMs are utilized in a cell where the device is functionalized by linking nucleic acid culture to deliver a particular agent and to monitor the effects building blocks to a therapeutic drug (e.g., Small molecule of Such an agent on cells or tissue cultures. Generally, the drug). DL-NAMs can be utilized in any method of the priros art [0177] In some embodiments, DL-NAMs are linked to vec where it is desired to transfect or genetically modify a cell. tors, such as derivatives of SV-40, adenovirus, retrovirus For example, in a method of screening different agents to derived DNA sequences and shuttle vectors derived from determine the mechanisms, by which Such compounds combinations of functional mammalian vectors and func induce cell differentiation, e.g., such as in studying effects on tional plasmids and phage DNA. Eukaryotic expression Vec stem cells. Methods of utilizing cell and tissue culture are tors are well known, e.g. such as those described by PJ known in theart, such as disclosed in U.S. Pat. Nos. 7.008,634 Southern and P Berg, J Mol Appl Genet. 1:327-341 (1982); (using cell growth substrates with tethered cell growth effec Subramini et al., Mol. Cell. Biol. 1:854-864 (1981), Kauf tor molecules); 6.972,195 (culturing potentially regenerative mann and Sharp, J. Mol. Biol. 159:601-621 (1982); Scahillet cells and functional tissue organs in vitro); 6,982,168 or al., PNASUSA 80:4654-4659 (1983) and Urlauband Chasin 6,962.980 (using cell culture to assay compounds for treating US 2010/013.6614 A1 Jun. 3, 2010 cancer); 6,902,881 (culturing techniques to identify sub taZone, were ultimately approved for the treatment of patients stances that mediate cell differentiation); 6,855,504 (cultur with Type II diabetes. Although these compounds were devel ing techniques for toxicology screening); or 6.846,625 (iden oped without an understanding of their molecular mechanism tifying validated target drug development using cell culture of action, by the early 90s evidence began to accumulate techniques). The DL-NAMS of the invention are readily linking the thiazolidinediones to the nuclear receptor PPAR adaptable to Such cell culturing techniques as would be evi gamma. It was ultimately demonstrated that these molecules dent to one of ordinary skill in the art. were high affinity ligands of PPAR-gamma and that they increased transcriptional activity of the receptor. Without Drugs of Use in the Invention wishing to be bound by theory, multiple lines of evidence now 0182. In some aspects of the invention, DL-NAMs of the indicate that the antidiabetic activities of the thiazolidinedi invention are linked to one or more drugs (e.g., FIGS. 18 and ones are mediated by their direct interaction with the receptor 20). DL-NAMs of the invention provide modular/anisotropic and the Subsequent modulation of PPAR-gamma target gene sites for linkage to one or more drugs. Furthermore, particular expression. DL-NAMs can be tailored to increase the number of one drug (0189 Thiazolidinediones of use in the methods of the or the number of a particular drug, through iterative rounds of invention include: (1) rosiglitaZone; (2) pioglitaZone; (3) tro conjugation. As such, different DL-NAMs comprise different glitazone; (4) netoglitazone (also known as MCC-555 or dosages. Of course, dosage can also be controlled by tempo isaglitazone or neoglitazone); and (5) 5-BTZD. ral regulation (e.g., number of administration, such as sys 0190. Other PPAR modulators of use in the invention temically, locally, epidermally, muscular injection, etc., as include modulators that have recently been the subject of well as frequency of administration in a given period of time. clinical trials: (1) Muraglitazar (PPARgamma and alpha ago 0183 Thus, the methods and compositions of the inven nist, Bristol-Myers/Merck); (2) Galida tesaglitazar (PPAR tion include the study and use of drugs, e.g., insulin sensitiz gamma and alpha agonist, AstraZeneca); (3) 677954 (PPAR ers, and include performing association studies for determin gamma, alpha, and delta agonist, GlaxoSmithKline); (4) ing genotypic and/or phenotypic traits associated with MBX-102 (PPARgamma partial agonist/antagonist, Metab responsiveness to drugs, e.g., insulin sensitizers, screening olex), (5) T131 (PPAR gamma selective modulator, Tularik/ individuals for predisposition to response to drugs, e.g., insu Amgen); (6) LYss (PPARgamma and alpha partial agonist, lin sensitizers, e.g., adverse response, and/or administering or Eli Lilly/Ligand); (7) LY929 (PPAR gammaandalphaagonist, not administering drugs, e.g., insulin sensitizers to the indi Eli Lilly/Ligand); and (8) PLX204 (PPAR gamma, alpha, and vidual based on such screening. The following relevant sec delta agonist, Plexxikon). See, e.g., BioCentury, Jun. 14, tions describe certain drugs of use in embodiments of the 2004. Further PPAR modulators include LY 519818, invention. Thus, in various embodiments, one or more of such L-783483, L-165461, and L-165041. drugs can be conjugated to DL-NAMs for delivery to a cell or 0191 Additionally, the non-thiazolidinediones that act as Subject. insulin-sensitizing agents include, but are not limited to: (1) 0184 Insulin Sensitizers JT-501 (JTT 501, PNU-1827, PNU-76-MET-0096, or PNU 0185. One class of drugs included in certain embodiments 182716: 4-(4-(2-(5-methyl-2-phenyl-oxazol-4-yl)ethoxy) of the invention is an insulin sensitizer. The term “insulin benzyl)isoxazolidine-3,5-dione: (2) KRP-297 (5-(2,4-di sensitizer, or “insulin sensitizing agent, as used herein, oXothiazolidin-5-ylmethyl)-2-methoxy-N-(4-(tri-fluorom refers to any agent capable of enhancing either secretion ofor, ethyl)benzyl)benzamide or 5-(2,4-dioxo-5-thiazolidinyl) more typically, tissue sensitivity to, insulin. Non-exclusive methyl)-2-methoxy-N-(4-(trifluoromethyl)phenyl)methyl) examples of insulin sensitizers include metformin, Sulfony benzamide); and (3) Farglitazar (L-tyrosine, N-(2- lureas, alpha glucosidase inhibitors and PPAR modulators, benzoylphenyl)-o-(2-(5-methyl-2-phenyl-4-oxazolyl)ethyl) including thiazolidinediones. Further examples of insulin O N-(2-benzoylphenyl)-O-(2-(5-methyl-2-phenyl-4-ox sensitizers are described below. azolyl)ethyl)-L-tyrosine, or (S)-2-(2-benzoylphenylamino)- 0186. The thiazolidinediones are examples of PPAR 3-(4-12-(5-methyl-2-phenyl-2-oxazo-4-Y) ethoxyphenyl) modulators, which are one class of insulin sensitizers. The propionic acid, or GW2570 or GI-262570). term “PPAR modulator, as used herein, refers to peroxisome (0192. Other agents have also been shown to have PPAR proliferator-activated receptor agonists, partial agonists, and modulator activity such as PPAR-gamma, SPPAR-gamma, antagonists. The modulator may, selectively or preferentially, and/or PPAR-alpha/delta agonist activity. Examples are: (1) affect PPAR alpha, PPARgamma, or both receptors. Typi AD 5075 (5-(4-(2-hydroxy-2-(5-methyl-2-phenyloxazol-4- cally, the modulator increases insulin sensitivity. According yl)ethoxy)benzyl)-thiaZolidine-2,4-dione); (2) R 119702 (or to one aspect, the modulator is a PPARgamma agonist. One C1 1037 or CS 011); (3) CLX-0940 (peroxisome proliferator PPARgamma agonist used in embodiments of the invention activated receptor alpha agonist/peroxisome proliferator-ac is 5-(6-(2-fluorobenzypoxy-2-naphthylmethyl-2,4-thia tivated receptor gamma agonist); (4) LR-90 (2.5.5-tris(4- Zolidinedione; (MCC-555 or “netoglitazone'). chlorophenyl)-1,3-dioxane-2-carboxylic acid, PPAR alpha/ 0187. Insulin Sensitizers PPAR Modulators gamma agonist); (5) CLX-0921 (PPAR gamma agonist); (6) 0188 One class of insulin sensitizers of the invention is CGP-52608 (PPAR agonist); (7) GW-409890 (PPAR ago PPAR modulators, and in particular PPAR-gamma modula nist); (8) GW-7845 (2C(S)-1-carboxy-2-(4-(2-(5-methyl-2- tors, e.g., PPAR-gamma agonists. PPAR modulators include phenyl-oxazol-4-Y)-ethoxy)-phenyl)-ethyamino)-benzoic the PPAR-alpha, PPAR-delta (also called PPAR-beta), and acid methyl ester, PPARagonist), (9) L-764406 (2-benzene PPAR-gamma agonists. Especially useful are the thiazo sulphonylmethyl-3-chloroquinoxaline, PPAR agonist); (10) lidinediones (TZDs), which were developed in the 70's and LG-101280 (PPARagonist); (11) LM-4156 (PPARagonist); 80s by screening newly synthesized compounds for their (12) Risarestat (CT-112, (+)-5-(3-ethoxy-4-(pentyloxy)phe ability to lower blood glucose in diabetic rodents. Three mol nyl-2,4-thiazolidinedione aldose reductase inhibitor); (13) ecules from this class, troglitaZone, rosiglitaZone, and piogli YM 440 (PPAR agonist); (14) AR-H049020 (PPARagonist); US 2010/013.6614 A1 Jun. 3, 2010 20

(15) GW 0072 ((+)-(2S.5S)-4-(4-(5-((dibenzy carbomoyl) Press, Oxford, 1986), in Immunoconiugates. Antibody Con methyl)-2-heptlyl-4-oxothiazolidin-3-ylbutyl)benzoic acid); jugates in Radioimaging and Therapy of Cancer (C.-W. (16) GW 409544 (GW-544 or GW-409544); (17) NN 2344 Vogel, ed., 3-300, Oxford University Press, New York, 1987), (DRF 2593); (18) NN 622 (DRF 2725); (19) AR-H039242 in Dillman, R. O. (CRC Critical Reviews in Oncology/He (AZ-242); (20) GW 9820 (fibrate); (21) GW 1929 (N-(2- matology 1:357. CRC Press, Inc., 1984), in Pastan et al.(Cell benzoylphenyl)-O-(2-(methyl-2-pyridinylamino) ethyl)-L- 47:641, 1986), in Vitetta et al. (Science 238:1098-1104, tyrosine, known as GW 2331, PPAR agonist); (22) SB 1987) and in Brady et al. (Int. J. Rad. Oncol. Biol. Phys. 219994 ((S)-4-(2-(2-benzoxazolylmethylamino)ethoxy)-al 13:1535-1544, 1987). Other examples of the use of immuno pha-(2.2.2-trifluoroethoxy)benzen epropanoic acid or 3-(4- conjugates for cancer and other forms of therapy have been (2-(N-(2-benzoxazolyl)-N-methylamino) ethoxy)phenyl)-2 disclosed, inter alia, in Goldenberg, U. S. Pat. Nos. 4.331,647, (S)-(2.2.2-trifluoroethoxy) propionic acid or benzenepro 4,348,376, 4,361,544, 4,468.457, 4,444,744, 4,460,459, panoic acid.4-(2-(2-benzoxazolylmethylamino) ethoxy)-al 4,460.561 and 4,624,846, and in Rowland, U.S. Pat. No. pha-(2.2.2-trifluoroethoxy)-, (alpha S)-, PPAR alpha/gamma 4,046.722, Rodwell et al., U.S. Pat. No. 4,671,958, and Shih agonist); (23) L-796449 (PPARalpha/gamma agonist); (24) et al., U.S. Pat. No. 4,699,784, the disclosures of all of which Fenofibrate (propanoic acid, 2-4-(4-chlorobenzoyl)phe are incorporated herein in their entireties by reference. noxy-2-methyl-, 1-methylethyl ester, known as TRICOR, 0.197 Other thiazolidinedione and non-thiazolidinedione LIPCOR, LIPANTIL, LIPIDIL MICRO PPARalpha ago insulin sensitizers of use in the invention are described in, nist); (25) GW-9578 (PPAR alpha agonist); (26) GW-2433 e.g., Leff and Reed (2002) Curr. Med. Chem. Imun., (PPARalpha/gamma agonist); (27) GW-0207 (PPAR gamma Endoc., & Metab. Agents 2:33-47: Reginato et al. (1998) J. agonist); (28) LG-100641 (PPAR gamma agonist); (29) Biol. Chem., 278 32679-32654; Way et al. (2001) J. Biol. LY-300512 (PPAR gammaagonist); (30) NID525-209 (NID Chem. 276 25651-25653; Shiraki et al. (2005).JBC Papers in 525); (31) VDO-52 (VDO-52); (32) LG 100754 (peroxisome Press, published on Feb. 4, 2005, as Manuscript proliferator-activated receptoragonist); (33) LY-510929 (per M500901200, and U.S. Pat. Nos. 4.703,052; 6,008,237; oxisome proliferator-activated receptor agonist); (34) bex 5,594,016; 6,838,442; 6,329,423: 5,965,589; 6,677,363; arotene (4-(1-(3,5,5,8,8-pentamethyl -5,6,7,8-tetrahydro-2- 4,572,912; 4,287,200; 4,340,605; 4,438,141; 4,444,779; naphthalenyl)ethenyl)benzoic acid, known as TARGRETIN, 4,572,912; 4,687,777; 4,725,610; 5,232,925; 5,002,953; TARGRETYN, TARGREXIN; also known as LGD 1069, LG 5,194,443; 5,260,445; 6,300,363; 6,034,110; and 6,541,493; 100069, LG 1069, LDG 1069, LG 69, RO 264455); and (35) U.S. patent application Publications 2002/0042441; 2004/ GW-1536 (PPARalpha/gamma agonist). 0198774 and 2003/0045553; EP Patent Nos. 0139421 and 0193 In some aspects of the invention, radioisotopes can 0332332; and PCT Publication Nos. WO 95/35314; WO be delivered via the implantable device of the invention. For 00/31055; WO 01/3640, all of which are incorporated by example, it is well known in the art that various methods of reference herein in their entirety. radionuclide therapy can be used for the treatment of cancer I0198) Netoglitazone and other pathological conditions, as described, e.g. in Har (0199. One thiazolidinedione PPAR modulator for use in bert, “Nuclear Medicine Therapy”, New York, Thieme Medi the methods of the invention is netoglitazone (5-(6-(2-fluo cal Publishers, 1987, pp. 1-340. A clinician experienced in robenzyl)oxy-2-naphthylmethyl-2,4-thiazolidinedione : these procedures will readily be able to adapt the implantable MCC-555). Structures and methods of preparation of netogli device described hereinto Such procedures to mitigate or treat taZone and various forms of netoglitaZone of use in the inven disease amenable to radioisotope therapy thereof. tion are described in, e.g., U.S. Pat. Nos. 5,594,016; 6.541, 0194 In some aspects the radio isotopes include but are 493: 6,541,493; 6,838,442; U.S. Patent Application No. not limited to isotopes and salts of isotopes with short half 2004/0198774 and 2003045553; PCT Publication Nos. WO life: such as Y-90, P-32, I-131, Au 198. Therefore in one 00/31055: WO 01/36401: WO 03/018010, and WO aspect of the invention, the implantable device can be utilized 00/73252: Japanese Patent Unexamined Publication to deliver radioisotopes. (KOKAL) Nos. (Hei) 6-247945/1994 and (Hei) 10-139768/ 0195 In some embodiments, DL-NAMs are linked to anti 1998: Japanese Patents 2001 172179 and 2003040877; and bodies alone or antibodies conjugated to radioisotopes. Reginatoetal. (1998).J. Biol. Chem. 273: 32679-32684; all of Therefore, antibodies can be directed to particular/specific which are incorporated by reference herein in their entirety. cellular epitope, thereby functioning as a targeting moiety. 0200. It has been reported that netoglitazone is more effi For example, many cellular epitopes are known in the art that cacious than pioglitaZone and troglitaZone in lowering are differentially expressed in different cell types (e.g., spe plasma glucose, insulin, and triglyceride levels and that it is cific organs, cancer/versus normal, diseased/versus non-dis about three-fold more potent than rosiglitazone. The activity eased). Therefore, in various embodiments, DL-NAMs can of netoglitaZone appears to be context-specific, as in some be targeted to particular cell types and to deliver atherapeutic cell types it behaves as a full agonist of PPAR-gamma and as drug. a partial agonist orantagonist in others. In addition, it appears 0196. It is also well known that radioisotopes, drugs, and to modulate PPAR-alpha and delta as well. See, e.g., U.S. toxins can be conjugated to antibodies or antibody fragments Patent Application Publication No. 2004/0198774. which specifically bind to markers which are produced by or 0201 Forms of Drugs associated with cancer cells, and that Such antibody conju 0202 Some compounds useful in the invention, including gates can be used to target the radioisotopes, drugs or toxins the TZD PPAR modulators such as netoglitazone, may have to tumor sites to enhance their therapeutic efficacy and mini one or more asymmetric carbon atoms in their structure. In mize side effects. Examples of these agents and methods are addition, Stereochemically pure isomeric forms of the com reviewed in Wawrzynczak and Thorpe (in Introduction to the pounds as well as their racemates can also be delivered using Cellular and Molecular Biology of Cancer, L. M. Franks and one or more matrix disclosed herein. Stereochemically pure N. M.Teich, eds, Chapter 18, pp. 378-410, Oxford University isomeric forms may be obtained by the application of art US 2010/013.6614 A1 Jun. 3, 2010

known principles. Diastereoisomers may be separated by 6,541,493; for example, the E form is described in PCT Pub physical separation methods such as fractional crystallization lished Application No. WO 01/36401. and chromatographic techniques, and enantiomers may be 0207 Some of the compounds described herein may exist separated from each other by the selective crystallization of with different points of attachment of hydrogen coupled with the diastereomeric salts with optically active acids or bases or double bond shifts, referred to as tautomers. An example is a by chiral chromatography. Pure Stereoisomers may also be carbonyl (e.g. a ketone) and its enol form, often known as prepared synthetically from appropriate stereochemically keto-enol tautomers. The individual tautomers as well as pure starting materials, or by using stereospecific reactions. mixtures thereof are encompassed within the invention. 0203 Some compounds useful in the invention may have 0208 Prodrugs are compounds that are converted to the various individual isomers, such as trans and cis, and various claimed compounds as they are being administered to a alpha and beta attachments (below and above the plane of the patient or after they have been administered to a patient. The drawing). In addition, where the processes for the preparation prodrugs are, compounds of this invention, and the active of the compounds according to the invention give rise to metabolites of the prodrugs are also compounds of the inven mixture of stereoisomers, these isomers may be separated by tion. conventional techniques such as preparative chromatography. 0209. Other agents useful in the methods of the invention The compounds may be prepared as a single stereoisomer or include, but are not limited to: in racemic formas a mixture of some possible stereoisomers. 0210 1. Biguanides, which decrease liverglucose produc The non-racemic forms may be obtained by either synthesis tion and increases the uptake of glucose. Examples include or resolution. The compounds may, for example, be resolved 0211 metformin such as: (1) 1,1-dimethylbiguanide (e.g., into their components enantiomers by standard techniques, Metformin-DepoMed, Metformin-Biovail Corporation, or such as the formation of diastereomeric pairs by salt forma METFORMINGR (metformin gastric retention polymer)); tion. The compounds may also be resolved by covalent link and (2) metformin hydrochloride (N,N-dimethylimidodicar age to a chiral auxiliary, followed by chromatographic sepa bonimidic diamide monohydrochloride, also known as LA ration and/or crystallographic separation, and removal of the 6023, BMS 207 150, GLUCOPHAGE, or GLUCOPHAGE chiral auxiliary. Alternatively, the compounds may be XR. resolved using chiral chromatography. Unless otherwise 0212 2. Alpha-glucosidase inhibitors, which inhibit noted the Scope of the bioactive agents, that can be included in alpha-glucosidase, and thereby delay the digestion of carbo the matrix(es) disclosed herein, is intended to cover all such hydrates. The undigested carbohydrates are Subsequently isomers or stereoisomers per se, as well as mixtures of cis and broken down in the gut, reducing the post-prandial glucose trans isomers, mixtures of diastereomers and racemic mix peak. Examples include, but are not limited to: (1) acarbose tures of enantiomers (optical isomers) as well. (D-glucose, O-4,6-dideoxy-4-(((1S-(1alpha,4alpha,5beta, 0204. In addition, compounds to be delivered by or 6alpha))-4.5,6-trihydroxy-3-(hydroxymethyll)–2-cyc-lo included in the matrixes of the invention may be prepared in hexen-1-yl)amino)-alpha-D-glucopyranosyl-(1-4)-O-alpha various polymorphic forms. For example, insulin sensitizers D-glucopyranosyl-(1-4)-, also known as AG-5421, Bay-g- of use in the invention can occur in polymorphic forms, and 542. BAY-g-542. GLUCOBAY, PRECOSE, GLUCOR, any or all of the polymorphic forms of these insulin sensitiz PRANDASE, GLUMIDA, or ASCAROSE); (2) Miglitol ers are contemplated for use in the invention. Polymorphism (3,4,5-piperidinetriol, 1-(2-hydroxyethyl)-2-(hydroxym in drugs may alter the stability, Solubility and dissolution rate ethyl)-, (2R (2alpha, 3beta, 4alpha, 5beta))-or (2R,3R.4R, of the drug and result in different therapeutic efficacy of the 5S)-1-(2-hydroxyethyl)-2-(hydroxymethyl-3,4,5-piperidin different polymorphic forms of a given drug. The term poly etriol, also known as BAY 1099, BAY M1099, BAY-m-1099, morphism is intended to include different physical forms, BAYGLITOL DIASTABOL. GLYSET MIGLIBAY. crystal forms, and crystalline/liquid crystalline/non-crystal MITOLBAY, PLUMAROL); (3) CKD-711 (0-4-deoxy-44(2. line (amorphous) forms. Polymorphism of compounds of 3-epoxy-3-hydroxymethyl-4,5,6-trihydro-xycyclohexane-1- therapeutic use has is significant, as evidenced by the obser yl) amino)-alpha-b-glucopyranosyl-(1-4)-alpha-D-glucopy Vations that many antibiotics, antibacterials, tranquilizers ranosyl-(1-4)-D-glucopyranose); (4) emiglitate (4-(2-((2R, etc., exhibit polymorphism and Some/one of the polymorphic 3R,4.R.5S)-3.45-trihydroxy-2-(hydroxymethyll)– 1 - forms of a given drug may exhibit Superior bioavailability and piperidinyl)ethoxy)benzoic acid ethyl ester, also known as consequently show much higher activity compared to other BAY o 1248 or MKC 542); (5) MOR 14 (3,4,5-piperidin polymorphs. For example, Sertraline, Frentizole, Ranitidine, etriol, 2-(hydroxymethyl)-1-methyl-, (2R-(2alpha, 3beta, Sulfathiazole, and Indomethacine are some of the pharma 4alpha, 5beta))-, also known as N-methyldeoxynojirimycin ceuticals that exhibit polymorphism. or N-methylmoranoline); and (6) Voglibose (3,4-dideoxy-4- 0205 Some embodiments of the invention include the use ((2-hydroxy-1-(hydroxymethyl)ethyl)amino)-2-C-(hy of netoglitaZone in one of its polymorphic forms. Netoglita droxymethyl)-D-epi-inositol or D-epi-lnosito 1,3,4-dideoxy Zone can be prepared in various polymorphic forms. Any 4-((2-hydroxy-1-(hydroxymethyl)ethyl)amino)-2-C- polymorphic forms of netoglitaZone known in the art may be (hydroxymethyl)-, also known as A 71100, AO 128, BASEN, used in the methods of the invention, either separately or in GLUSTAT, VOGLISTAT combination. Thus, the methods of the invention include 0213 3. Insulins include regular or short-acting, interme association studies using any or all of the polymorphic forms diate-acting, and long-acting insulins, injectable, non-inject ofnetoglitaZone, as well as screening and treatment using any able or inhaled insulin, transderamal insulin, tissue selective or all of the polymorphic forms of netoglitaZone, composi insulin, glucophosphokinin (D-chiroinositol), insulin ana tions and kits based on these forms, and the like. logues such as insulin molecules with minor differences in the 0206 Polymorphic forms of netoglitazone include the A, natural amino acid sequence and Small molecule mimics of B, C, D, E and amorphous crystal forms described in PCT insulin (insulin mimetics), and endoSome modulators. Published Application No. WO 01/36401 and in U.S. Pat. No. Examples include, but are not limited to: (1) Biota; (2) LP

US 2010/013.6614 A1 Jun. 3, 2010 24

0230 Mechanistic Classes of Drugs administration. For example, insulin and insulin analogs may 0231. One non-exclusive exemplary classes of drugs for be formulated for administration by injection, nasal spray, which genotyping and association studies with one member transdermal, oral or inhalation routes. Each type of formula may be used to predict effects of another member include, tion can have unique profiles of responses and associated mechanistic classes of drugs used in the treatment of diabetes genetic variations. An example of classifications of Such (including PPAR modulators). This class of drugs also illus drugs by mechanism, together with representative members trates how drugs can also be subclassed by, e.g., mode of of the mechanistic classes, is given in the table below.

TABLE 6 Classes of Drugs for Treatment of Diabetes Class Mechanism of Action Examples Peroxisome Target PPAR-gamma or PPAR-gamma and -alpha (see below). RosiglitaZone, PioglitaZone, Proliferator PPAR are nuclear receptors that help regulate glucose and lipid BalaglitaZone, see also Activated Receptor metabolism. Activation of PPAR-gamma improves insulin others described herein (PPAR) Agonists sensitivity and thus improves glycemic control. Dual-Action Act on both PPAR-gamma and PPAR-alpha. PPAR-alpha TAK-559, Muraglitazar, Peroxisome activation has effects on cellular uptake of fatty acids and their Tesaglitazar, NetoglitazOne, Proliferator oxidation, and on lipoprotein metabolism. May also act to reduce See also others described Activated Receptor inflammatory response in vascular endothelial cells. herein Agonists Biguanidines Complete mechanism is not known. Reduces gluconeogenesis in Metformin, Metformin GR the liver by inhibiting glucose-6-phosphatase. Sulfonylureas Induce insulin Secretion by binding to cellular receptors that Glimepride, cause membrane depolarization and insulin exocytosis. Glyburide? glibenclamide, Glipizide, Gliclazide. Tobutamide Insulin and Insulin Supplements endogenous insulin. Insulin analogs have a variety Insulin lispro, Insulin aspart, Analogs (Injectable, of amino acid changes and have altered onset of action and Insulin glargine, Exubera, Inhaled, Oral, duration of action, as well as other properties, compared to native AERX Insulin Diabetes Transdermal, insulin. Inhaled insulin is absorbed through the alveoli. Spray Management System, HIM Intranasal) oral insulin is absorbed by the buccal mucosa and intranasal 2, Oaralin, Insulin detemir, through the nasal mucosa. Transdermal insulin is absorbed Insulin glulisine through the skin. Meglitinides Are thought to bind to a nonsulfonylurea beta cell receptor and Repaglinide, Nateglinide, act to cause insulin secretion by mechanism similar to Mitiglinide sulfonylureas Alpha-Glucosidase Inhibit carbohydrate digestion. Act at brush border of intestinal Acarbose, Miglitol, Inhibitors epithelium. Voglibose Glucagon-Like Diabetic patients may lack native GLP-1, and anlalogs act as Exenatide, Exenatide LAR, Peptide(GLP)-1 Substitutes. GLP-1 is an intestinal peptide hormone that induces Liraglutide, ZP 10, Analogs glucose-dependent insulin secretion, controls gastric emptying, BN51077, inhibits appetite, and modulates secretion of glucagon and Somatostatin. Dipeptidyl Peptidase Inhibit DPP-IV, a ubiquitous enzyme that cleaves and inactivates LAF-237, p-32/98, MK (DPP)-IV Inhibitors GLP-1, thus inhibition of DPP-IV increases GLP-1 activity 431, P3298, NVP LAF 237, Pancreatic Lipase Inhibits lipases, thus inhibiting uptake of dietary fat. This causes Orlistat Inhibitors weight loss, improves insulin sensitivity and lowers hyperglycemia. Amylin Analogs Act to augment amylin, which acts with insulin by slowing Pramlintide glucose absorption from the gut and slows after-meal glucose release from liver. Dopamine D2 Thought to act to alleviate abnormal daily variations in central Bromocriptine receptor agonists neuroendocrine activity that can contribute to metabolic and immune system disordered. ImmunOSuppressants Suppress autoimmune response thought to be implicated in Daclizumab, NBI 6024, Type I and possibly Type II diabetes. Example: Humanized TRX-TolerRx, OKT3 monoclonal antibody that recognizes and inhibits the alpha gamma-1-ala-ala subunit of IL-2 receptors; humanized Mab that binds to T cell CD3 receptor to block function of T-effector cells that attack the body and cause autoimmune disease Insulin-like growth Recombinant protein complex of insulin-like growth factor-1 and Somatomedin-1 binding factor-1 agonists binding protein-3; regulates the delivery of somatomedin to target protein 3 tissues. Reduces insulitis severity and beta cell destruction Insulin sensitizers Insulin sensitizers, generally orally active S15261, Dexlipotam, CLX 0901, R 483, TAK 654 Growth hormone Mimic the action of native GHRF TH9507, SOM 230 releasing factor agonists Glucagon antagonists Inhibit glucagon action, stimulating insulin production and Liraglutide, NN 2501 Secretion, resulting in lower postprandial glucose levels Diabetes type 1 Prevents destruction of pancreatic beta cells that occurs in type 1 Q-Vax, Damyd vaccine vaccine diabetes

US 2010/013.6614 A1 Jun. 3, 2010 28

0235. In other embodiments, mechanistic classes of drugs 0238 Sedative-Hypnotic Drugs, which include drugs that used in the treatment of Parkinson's disease are used in con bind to the GABAA receptor such as the benzodiazepines junction with a method or composition of the invention. (including alprazolam, chlordiazepoxide, cloraZepate, clon These classes include dopamine precursors, dopamine ago azepam, diazepam, estaZolam, flurazepam, halazepam, nists, COMT inhibitors, MAO-B inhibitors, antiglutameter lorazepam, midazolam, oxazepam, quazepam, temazepam, gic agents, anticholinergic agents, mixed dopaminergic triazolam), the barbiturates (such as amobarbital, pentobar agents, adenosine A2a antagonists, alpha-2 adrenergic bital, phenobarbital, secobarbita), and non-benzodiazepines antagonists, antiapoptotic agents, growth factor Stimulators, (such as Zolpidem and Zaleplon), as well as the benzodiaz and cell replacements. An example of classification of Such epine antagonists (such as flumazenil). Other Sedative-hyp drugs by mechanism, together with representative members notic drugs appear to work through non-GABA-ergic mecha of the mechanistic classes, is given the Table below. nisms such as through interaction with serotonin and

TABLE 10 Classes of Drugs for Treatment of Parkinson's Disease Class Mechanism of Action Examples Dopamine Precursors Act as precursors in the synthesis of dopamine, the Levodopa, Levodopa neurotransmitter that is depleted in Parkinson's Disease. Usually carbidopa, Levodopa administered in combination with an inhibitor of the carboxylase benserazide, Etilevodopa, enzyme that metabolizes levodopa. Some (e.g., Duodopa) are Duodopa given by infusion, e.g., intraduodenal infusion Dopamine Agonists Mimic natural dopamine by directly stimulating striatal dopamine Bromocriptine, Cabergoline, receptors. May be subclassed by which of the five known Lisuride, Pergolide, dopamine receptor Subtypes the drug activates; generally most Pramipexole, Ropinirole, effective are those that activate receptors the in the D2 receptor TalipeXole, Apomorphine, family (specifically D2 and D3 receptors). Some are formulated Dihydroergocryptine, for more controlled release or transdermal delivery. Lisuride, Piribedil, Talipexole, Rotigotin CDS, Sumanirole, SLV-308 COMT Inhibitors Inhibits COMT, he second major enzyme that metabolized Entacapone, Tolcapone, levodopa. Entacapone-Levodopa Carbidopa fixed combination, MAO-B Inhibitors MAO-B metabolizes dopamine, and inhibitors of MAO-B thus Sellegiline, Rasagiline, prolong dopamines half-life Safinamide Antiglutamatergic Block glutamate release. Reduce levodopa-induced dyskinesia Amantadine, Budipine, Agents Talampanel, Zonisamide Anticholinergic Thought to inhibit excessive cholinergic activity that Trilhexyphenidyll, Agents accompanies dopamine deficiency Benztropine, Biperiden Mixed Dopaminergic Act on several neurotransmitter systems, both dopaminergic and NS-2330, Sarizotan Agents nondopaminergic. Adenosine A2a Adenosine A2 antagonize dopamine receptors and are found in Istradefylline antagonists conjunction with dopamine receptors. Antagonists of these receptors may enhance the activity of dopamine receptors. Alpha-2 Adrenergic Not known. Yohimbine, Idazoxan, Antagonists Fipamezole Antiapoptotic Agents Can slow the death of cells associated with the neurodegenerative CEP-1347, TCH-346 process of Parkinson's disease. Growth Factor Promote the Survival and growth of dopaminergic cells. GPI-1485, Glial-cell-line Stimulators derived Neurotrophic Factor, SR-57667, PYM SOO28 Cell Replacement Replace damaged neurons with health neurons. Spheramine Therapy

0236. The above classifications are exemplary only. It will dopaminergic receptors, and include buspirone, isapirone, be appreciated that a drug class need not be restricted to drugs geprinone, and tandospirone. Older drugs work through used in the treatment of a single disease, but that a given mechanisms that are not clearly elucidated, and include chlo mechanistic class may have members useful in the treatment ral hydrate, ethchlorVynol, meprobamate, and paraldehyde. of a number of diseases. For example, MAO-B inhibitors are useful in the treatment of both Parkinson's disease and 0239. In some embodiments, sedative-hypnotic drugs that depression; as another example, statins are useful in the treat interact with the GABA receptor, such as benzodiazepines ment of dyslipidemias but are also being found to have more and non-benzodiazepines, are further classified as to which general use in diseases where inflammation plays a major subunit or subunits of the GABAA receptor that they interact role, e.g., multiple Sclerosis and other diseases. 0237 Further classifications of drugs by mechanism are with, e.g., thea (which is further classified into six Subtypes, known in the art; often these classifications may be further including C-1.2.3, and 5), B (further classified as four differ classified by structure. Non-exclusive examples of drug ent types), Y (three different types), Ö, e, , p, etc. Such a classes useful in the methods and compositions of the inven classification can allow further refinement of associations tion, and representative members of these classes, include: between genetic variation and responsiveness to a given seda US 2010/013.6614 A1 Jun. 3, 2010 29 tive-hypnotic that interacts with a particular Subclass, and emedastine, fexofenadine, hydroxyzine, ketotifen, levo predictions for a new sedative-hypnotic that interacts with the cabastine, loratadine, meclizine, olopatadine, phenindamine, same Subclass of receptors. and promoathazine. 0240 Opioid analgesics and antagonists act on the opioid 0243 Drugs used in asthma include sympatheticomimet receptor. The majority of currently available opioid analge ics (used as “relievers,” or bronchodilators) such as albuterol, sics act primarily at the L opioid receptor. However, interac albuterol/lpratropium, bitolterol, ephedrine, epinephrine, for tions, also occur with the 8 and K receptors. Similar to the moterol, isoetharine, isoproterenol, levalbuterol, metaproter sedative-hypnotics, in Some embodiments opioid analgesics enol, pirbuterol, salmeterol, salmeterol/fluticasone, terbuta line; aerosol corticosteroids (used as “controllers.” or are further classed as to subtypes of receptors at which they antiinflammatory agents) such as beclomethasone, budes primarily interact, thus allowing further refinement of the onide, flunisolide, fluticasone, fluticasone/salmeterol, triam association between drug response and genetic variation, and cinolone; leukotriene inhibitors such as montelukast, higher predictability for a new drug, based on which receptor Zafirlukast, Zileuton; cormolyn Sodium and nedocromil (s) it interacts with. Opioid analgesics include alfentanil, Sodium; methylxanthines Such as aminophylline, theophyl bupremorphine, butorphanol, codeine, dezocine, fentanyl, linem dyphylline, OXtriphylline, pentoxifylline; antimuscar hydromorphone, levomethadyl acetate, levorphanol, meperi inic drugs such as ipratropium; and antibodies such as oma dine, methadone, morphine sulfate, nalbuphine, oxycodone, liZumab. oxymorphone, pentazocine, propoxyphene, remifentanil, 0244 Erectile dysfunction drugs include c0MP enhanc Sufentanil, tramadol; analgesic combinations such as erS Such as sildenafil (Viagra), tadalafil. Vardenafil, and codeinefacetaminophen, codeine/aspirin, hydrocodone?ac alprostadil, and dopamine releasers such as apomorphine etaminophen, hydrocodone/ibuprofen, oxycodone/acetami 0245] Drugs used in the treatment of gastrointestinal dis nophen, oxycodone/aspirin, propoxyphene/aspirin or ease act by a number of mechanisms. Drugs that counteract acetaminophen. Opioid antagonists include nalmefene, acidity (antacids) include aluminum hydroxide gel, calcium naloxone, naltrexone. Antitussives include codeine, dex carbonate, combination aluminum hydroxide and magne tromethorphan. sium hydroxide preparation. Drugs that act as proton pump 0241. Nonsteroidal anti-inflammatory drugs act primarily inhibitors include esomeprazole, lanSoprazole, pantoprazole, through inhibition of the synthesis of prostaglandins, e.g., and rabeprazole. H2 histamine blockers include cimetidine, through inhibition of COX-1, COX-2, or both. Older famotidine, nizatidine, ranitidine. Anticholinergic drugs include atropine, belladonna alkaloids tincture, dicyclomine, NSAIDS (e.g., salicylates) tend to be non-selective as to the glycopyrrolate, I hyoscyamine, meth scopolamine, propan type of COX inhibited, whereas newer drugs are quite selec theline, scopolamine, tridihexethyl. Mucosal protective tive (e.g., the COX-2 inhibitors). Non-selective COX inhibi agents include misoprostol. Sucralfate. Digestive enzymes tors include aspirin, acetylsalicylic acid, choline Salicylate, include pancrelipase. Drugs for motility disorders and anti diclofenac, etodolac, fenoprofen, flurbiprofen, ibuprofen, emetics include alosetron, cisapride, dolasetron, dronabinol, indomethacin, ketoprofen, ketorolac, magnesium salicylate, granisetron, metoclopramide, ondansetron, prochlorpera meclofenamate, mefenamic acid, nabumetone, naproxen, Zine, tegaserod. Antiinflammatory drugs used in gastrointes oxaprozin, phenylbutazone, piroxicam, salsalate, salicylsali tinal disease include balsalazide, budesonide, hydrocorti cylic acid, Sodium salicylate, Sodium thiosalicylate, Sulindac, SOne, mesalamine, methylprednisone, olsalazine, tenoxicam, tiaproven, azapropaZone, carprofen, and tol Sulfasalazine, infliximab. Antidiarrheal drugs include bis metin. Selective COX-2 inhibitors include celecoxib, etrori muth subsalicylate, difenoxin, diphenoxylate, kaolin/pectin, coxib, meloxicam, rofecoxib, and Valdecoxib. loperamide. Laxative drugs include bisacodyl, cascara 0242. Histamine agonists and antagonists are classified sagrada, castor oil, docusate, glycerin liquid, lactulose, mag according to receptor Subtype. H1 agonists or partial agonists nesium hydroxide milk of magnesia, Epson Salt, methyl include 2-(m-fluorophenyl)-histamine and antagonists cellulose, mineral oil, polycarbophpil, polyethylene glycol include chlorpheniramine, scopolamine, mepyramine, ter electrolyte Solution, psyllium, Sienna. Drugs that dissolve fenadine, astemizole, and triprolidine; further antagonists gallstones include monoctanoin, ursodiol. (which may be further classified by their chemical structures) 0246 Cholinoceptor-activating drugs, which act by acti include the ethanolamines carbinoxamine, dimenhydrinate, Vating muscarinic and/or nicotinic receptors include esters of diphenhydramine, and doxylamine; the ethylaminediamines choline (e.g., acetylcholine, metacholine, carbamic acid, car pyrilamine and tripelennamine; the piperazine derivatives bachol, and bethanechol) and alkaloids (e.g., muscarine, pilo dydroxy Zine, cyclizine, feXofenadine and meclizine; the carpine, lobeline, and nicotine); cholinesterase-inhibiting alkylamines brompheniramine and chlorpheniramine; and drugs which typically act on the active site of cholinesterase miscellaneous antagonists cyproheptadine, loratadine, cetriz include alcohols bearing a quaternary ammonium group (e.g., ine. H2 agonists include dimaprit, impromidine, and edrophonium), carbamates and related agents (e.g., neostig amthamine; and antagonists (useful in the treatment of gastric mine, physostigmine, pyridostigmine, ambenonium, and acid secretion) include cimetidine, ranitidine, nizatidine, and demercarium), and organic derivatives of phosphoric acid famotidine; H3 agonists include R-alpha-methylhistamine, (e.g... echothiophate, soman, parthion, malathion); echoli imetit, and immepip and antagonists include thioperamide, noceptor-blocking drugs typically act as antagonists to nico iodophenpropit, and clobenpropit; and H4 agonists include tinic receptors (further classified as ganglion-blockers. Such clobenpropit, imetit, and clozapine and antagonists include as hexamethonium, mecmylamine, teteraethylammonium, thioperamide. Available preparations include the H1 blockers and trimethaphan; and neuromuscular junction blockers, see azelastine, brompheniramine, buclizine, carbinoxamine, skeletal muscle relaxants) orantagonists to muscarinic recep cetrizine, chlorpheniramine, clemastine, cyclizine, cyprohep tors (e.g. atropine, propantheline, glycopyrrolate, piren tadine, desloratidine, dimenhydrinate, diphenhydramine, Zepine, dicyclomine, tropicamide, ipatropium, banZtropine, US 2010/013.6614 A1 Jun. 3, 2010 30 gallamine, methooctramine, AF-DX 116, telenzipine, triheX ganglion-blocking drugs, Suprea; adrenergic neuron-block yphenidyl, darifenacin, Scopolamine, homatropine, cyclo ing agents such as gunethidine, gunadrel, bethanidine, pentolate, anisotropine, clidinium, isopropamide, mepen debrisoquin, and reserpine; adrenoceptor antagonists such as Zolate, methScopolamine, oxyphenonium, propantheline, propanolol, metoprolol, nadolol, carteolol, atenolol, betaX oxybutynin, oxyphencyclimine, propiverine, tolterodine, olol, bisoprolol, pindolol, acebutolol, and penbutolol, labe tridihexethyl), which can be further subclassed as to which talol, carvedilol, esmolol, paZosin, phentolamine and phe muscarinic receptor is the primary site of the effect, e.g., M1, noxybenzamine; vasodilators such as hydralzaine, minoxidil, M2, M3, M4, or M5, allowing greater predictability for an Sodium nitroprusside, diazoxide, fenoldopam, and calcium association between a genetic variation and a response for a channel blockers (e.g. Verapamil, diltiazem, amilopidine, new drug based on its primary site of effect. Available prepa felopidine, isradipine, nicardipine, nifedipine, and nisol rations of antimuscarinic drugs include but are not limited to atropine; beladonna alkaloids, extract, or tincture; clidinium; dipine); ACE-inhibitors such as captropril, enalapril, lisino cyclopentolate; dicyclomine; flavoxate; glycopyrrolate; pril, benazepril, fosinopril, moexipril, perindopril, duinapril, homatropine; 1-hysocyamine, ipratropium; mepenZolate: ramipril, and trandolapril; angiotensin receptor blocking methantheline: methscopolamine: Oxybtynin; prpantehline: agents such as losartan, Valsartan, candesartan, eprosartan, scopolamine; tolterodine; tridihexethyl:, tropicamide. Avail irbesartan, and telmisartan. Preparations available include: able preparations of ganglion blockers include mecamy beta adrenoceptor blockers acebutolol, atenolol, betaxolol, lamine and trimethaphan. Available cholinesterase regenera bisoprolol, carteolol, carvedilol, exmolol, labetalol, meto tors include pralidoxime. prolol nadolol pembutolol, pindolol, propanolol, timolol: 0247 Adrenoceptor-activating drugs and other sympatho centrally acting sympathoplegic drugs clonidine, gunabenz, mimetic drugs may be classified according to the receptor or guanfacine, methyldopa; postganglionic sympatheic nerve receptors that they activate, e.g., alpha-one type (including terminal blockers gunadrel, guanethidine, and reserpine; Subtypes A, B, D), alpha-two type (including Subtypes A, B, alpha one selective adrenoceptor blockers doxazosin, pra and C), beta type (including Subtypes 1, 2, and 3), and dopam Zosin, terazosin: ganglion-blocking agent mecamylamine: ine type (including Subtypes 1,2,3,4, and 5. Exemplary drugs Vasodilators diazoxide, fenoldopam, hydralazine, minoxidil, include epinephrine, norepinephrine, phenylephrine, meth nitroprusside; calcium channel blockers amlodipine, dilt oxamine, milodrine, ephedrine, XylometaZoline, amphet iazem, felodipine, isradipine, nicardipine, nisoldipine, nife amine, methamphetamine, phenmetrazine, methylphenidate, dipine, verapamil; ACE inhibitors benazepril, captopril, phenylpropanolamine, methylnorepinephrine, dobutamine, enalapril, fosinopril, lisinopril, moexipril, perindopril, clonidine, BHT920, oxymetazoline, isoproterenol, pro quinapril, ramipril, and trandolapril; and angiotensin receptor caterol, terbutaline, metaproterenol, albuterol, ritodrine, blockers candesartan, eprosartan, irbeartan, losartan, olimis BRL37344, dopamine, fenoldopam, bromocriptine, quin artan, telmisartan, and Valsartan. pirol, dexmedetomidine, tyramine, cocaine (dopamine 0250 Vasodilators used in angina pectoris include nitric reuptake inhibitor), apraclonidine, brimonidine, ritodrine, oxide releasing drugs such as nitric and nitrous acid esters of terbutaline, and modafinil. Available preparations include polyalcohols Such as nitroglycerin, isorbide dinitrate, amyl amphetamine, apraclonidine, brimonidine, dexmedetomi nitrite, and isosorbide mononitrate; calcium channel blockers dine, dexmthylphenidate, dextroamphetamine, dipivefrin, Such as amlodipine, felodipine, isradipine, nicardipine, nife dobutamine, dopamine, ephedrine, epinephrine, fenoldopam, dipine, nimodipine, nisoldipine, nitrendipine, bepridil, dilt hydroxyamphetamine, isoproterenol, mephentermine, met iazem, and Verapamil; and beta-adrenoceptor-blocking drugs araminol, methamphetamine, methoxamine, methylpheni (see above). Available preparations include: nitrates and date, midodrine, modafinil, naphazoline, norepinephrine, nitrites amyl nitrite, isosorbide dinitrate, isosorbide mononi OXymetZoline, pemoine, phendimetraZine, phenylephrine, trate, nitroglycerin; calcium channel blockers amlodipine pseudoephedrine, tetrahydrozoline, and Xylometaoline. bepridil, diltiazem, felodipine, isradipine, nicardipine, nife 0248 Adrenoceptor antagonist drugs may be classified by dipine, nimodipine, nisoldipine, and Verapamil; and beta receptor Type In the same manner as adrenoceptor agonists, blockers acebutolol, atenolol, betaxolol, bisoprolol, car and include tolazoline, dibenamine, prazosin, terazosin, doX teolol, carvedilol, esmolol, labetolol, levobunolol, azosin, phenoxybenzamine, phentolamine, rauwoscine, metilproanolol, nadolol, pembutolol, pinolol, propanolol, yohimbine, labetalol, carvedilol, metoprololol, acebutolol, sotalol, timolol. alprenolol, atenolol, betaxolol, celiprolol, esmolol, pro 0251 Drugs used in heart failure include cardiac glyco panolol, carteolol, penbutolol, pindolol, timolol, butoxamine, Sides Such as digoxin; phosphodiesterase inhibitors Such as ergotamine, dihydroergotamine, tamulosin, alfuZosin, inmrinone and milrinone; beta adrenoceptor stimulant Such indoramin, urapidil bisoprolol nadolol, sotalol, Oxpenolol, as those described: diuretics as discussed below; ACE inhibi bopindolol, medroxalol, and bucindolol. Available prepara tors such as those discussed above; drugs that inhibit both tions include: alpha blockers doxazosin, phenoxybenzamine, ACE and neutral endopeptidase Such as omaprtrilat, vasodi phentolamine, prazosin, tamsulosin, teraZosin, and tolazo lators such as synthetic brain natriuretic peptide (nesiritide) line; and beta blockers acebutolol, atenolol, betaxolol, biso and bosentan; beta adrenoceptor blockers such as those prolol carteolol carvedilol, esmolol, labetolol, levobunolol described above. Available preparations include: digitalis metilproanolol nadolol, pembutolol, pinolol, propanolol, digoxin, digitalis antibody digoxin immune Fab; Sympatho Sotalol, timolol; and synthesis inhibitor metyrosine. mimetics dobutamine and dopamine; ACE inhibitors capto 0249 Antihypertensive agents include drugs that work by pril, enalapril, fosinopril, lisinopril, duinapril, ramipril, and a variety of mechanisms and thus overlap with other classifi trandolapril; angiotensin receptor blockers candesartan, cations. Agents can include diuretics such as thiazide diuret wprosartan, irbesartan, losartan, olmesartan, telmisartan, and ics, and potassium sparing diurietics; drugs that act on the valsartan; beta blockers bisoprolol, carvedilol, and meto central nervous system such as methyldopa and clonidine; prolol. US 2010/013.6614 A1 Jun. 3, 2010

0252 Cardiac arrhythmia drugs include drugs that act by epoprostenol, latanoprost, misoprostol, monteleukast, blocking Sodium channels such as quinidine, amiodaron, travaprost, treprostinil, unoprostone, Zafirleukast, Zileuton. disoprymide, flecamide, lidocaine, mexiletine, morcizine, Further eicosanoid modulators are discussed elsewhere procainamide, propafeneone, and tocamide; beta-adrenocep herein as nonsteroidal antiinflammatory drugs (NSAIDs) tor-blocking drugs such as propanolol, esmolol, and Sotalol; drugs that prolong the effective refractory period by prolong 0258 Drugs for the treatment of acute alcohol withdrawal ing the action potential Such as amiodarone, bretylium, include diazepam, lorazepam, oxazepam, thiamine, drugs for Sotalol, dolfetilide, and ibutilide; calcium channel blockers prevention of alcohol abuse include disulfuram, naltrexone; Such as Verapamil, diltizem, and bepridil; and miscellaneous and drugs for the treatment of acute methanol or ethylene agents such as adenosine, digitalis, magnesium, and potas glycol poisoning include ethanol, fomepizole. sium. Available preparations include: the sodium channel 0259 Antiseizure drugs include carbamazepine, clon blockers disopryamide, flecamide, lidocaine, miexiletine, azepam, clorazepate dipotassium, diazepam, ethosuximide, moricizine, procainamide, propafenone, quinidine Sulfate, ethotoin, felbamate, fosphenyloin, gabapentin, llamotrigine, quinidine gluconate, and quinidine polygalacturonate; the levetiracetam, lorazepam, mephenyloin, mephobarbital, oxy beta blockers acebutolol, esmolol, and propranolol; the action carbazepine, pentobarbital Sodium, phenobarbital, pheny potential-prolonging agents amiodarone, bretylium, dolfetil loin, primidone, tiagabine, topiramate, trimethadione, Valp ide, ibutilide, and sotalol; the calcium channel blockers bepri roic acid. dil, diltiazem, and Verapamil; and adenosine and magnesium 0260 General anesthetics include desflurane, dexmedeto sulfate. midine, diazepam, droperidol, enflurane, etomidate, hal 0253) Diuretic agents include drugs that act as carbonic othane, isoflurane, ketamine, lorazepam, methohexital, meth anhydrase inhibitors such as acetazoloamide, dichlorphena oxyflurane, midazolam, nitrous oxide, propofol, sevoflurane, mide, methazolamide; loop diuretics Such as furosemide, thiopental. bumetanide, torsemide, ethacrynic acid, and mercurial diuretics; drugs that inhibit NaCl transport in the distal con 0261 Local anesthetics include articaine, benzocaine, Voluted tubule and, in some cases, also act as carbonic anhy bupivacaine, butamben picrate, chloroprocaine, cocaine, drase inhibitors, such as bendroflumethiazide, benzthiazide, dibucaine, dyclonine, levobupivacaine, lidocaine, lidocaine chlorothiazide, chlorthalidone, hydrochlorothiazide, hydrof and etidocaine eutectic mixture, mepivacaine, pramoxine, lumethiazide, indapamide, methyclothiazide, metolaZone, prilocalne, procaine, proparacaine, ropivacaine, tetracaine. polythiazide, quinethazone, and trichlormethazide; potas 0262 Skeletal muscle relaxants include neuromuscular sium-sparing diuretics such as spironolactone, triamterene, blocking drugs such as atracurium, cisatracurium, doxacu eplerenone, and amiloride: osmotic diuretics such as manni rium, metocurine, mivacurium, pancuronium, pipecuronium, tol; antidiuretic hormone agonists such as Vasopressin and rocuronium, succinylcholine, tubocurarine, Vecuronium; desmopressin; antidiuretic hormone antagonists Such muscle relaxants (spasmolytics) such as baclofen, botulinum aslithium and demeclocycline. Available preparations toxin type A, botulinum toxin type B, carisoprodol, chorphen include actetazolamide, amiloride, bendroflumethiazide, esin, chlorzoxazone, cyclobenzaprine, dantrolene, diazepam, benzthiazide, brinZolamide, bumetanide, chlorothiazide, gabapentin, metaxalone, methocarbamol, orphenadrine, rilu chlorthalidone, demeclocycline, dichlorphenamide, dorZola Zole, and tizanidine. mide, eplerenone, ethacrynic acid, furosemide, hydrochlo I0263. Antipsychotic agents include aripiprazole, chlor rothiazide, hydroflumethiazide, indapamide, mannitol, met promazine, clozapine, fluphenazine, fluiphenazine esters, hazolamide, methyclothiazide, metolazione, polythiazide, haloperidol, haloperidol ester, loxapine, mesoridazine, quinethaZone, apironolactone, torsemide; triamterene, and molindone, olanzapine, perphenazine, pimozide, prochlor trichlormethiazide. perazine, promazine, quetiapine, risperidone, thioridazine, 0254 Serotonin and drugs that affect serotonin include thiothixene, trifluoperazine, triflupromazine, Ziprasidone; serotoninagonists such as fenfluramine and dexfenfluramine, mood stabilizers include carbamazepine, divalproex, lithium buspirone, Sumatriptan, cisapride, tegaserod; seratonin carbonate, and valproic acid. antagonists p-chlorophenylalanine and p-chloroamphet 0264. Agents used in anemias include hematopoietic amine, and reserpine; and the serotonin receptor antagonists growth factors such as darbopoetin alfa, deferoxamine, epo phenoxybenzamine, cyproheptadine, ketanserin, ritanserin, etin alfa (erythropoetin, epo), filgrastim (G-CSF), folic acid, and ondansetron; serotonin reuptake inhibitors are described iron, oprelvekin (interleukin 11), pegfilgrastim, sargra elsewhere herein. Serotonin receptor agonists include almot mostim (GM-CSF), vitamin B12. riptan, eletriptan, frovatriptan, naratriptan, rizatriptan, 0265 Disease-modifying antirheumatic drugs include Sumatriptan, and Zolmitriptan. anakinra, adalimumab, auranofin, aurothioglucose, eta ner 0255 Ergot alkaloids are useful in the treatment of, e.g., cept, gold sodium thiomalate, hydroxychloroquine, inflix migraine headache, and act on a variety of targets, including imab, leflunomide, methotreXate, penicillamine, sulfasala alpha adrenoceptors, serotonin receptors, and dopamine Zine. Drugs used in gout include allopurinol, colchicine, receptors. They include bromocriptine, cabergoline, per probenecid, sulfinpyraZone. golide, ergonovine, ergotamine, lysergic acid diethylamide, 0266 Drugs used in disorders of coagulation include and methysergide. Available preparations include dihydroer abciximab, alteplase recombinant, aminocaproic acid, anis gotamine, ergonovine, ergotamine, ergotamine tartrate, and indione, antihemophilic factor [factor VIII, AHF), anti-in methylergonovine. hibitor coagulant complex, antithrombin III, aprotinin, arga 0256 Vasoactive Peptides include aprepitant, bosentan. troban, bivalirudin, cilostazol, clopidogrel, coagulation 0257 Eicosanoids include prostaglandins, thomboxanes, factor VIIa recombinant, dalteparin, danaparoid, dipy and leukotrienes. Eicosanoid modulator drugs include alpros ridamole, enoXaparin, eptifibatide, Factor VIIa, Factor VIII, tadil, bimatoprost, carboprost tromethamine, dinoprostone, Factor IX, fondaparinux, heparin sodium, lepirudin, phytona US 2010/013.6614 A1 Jun. 3, 2010 32 dione K1, protamine, reteplase, Streptokinase, tenecteplase, calcitonin-Salmon, etidronate, gallium nitrate, pamidronate, ticlopidine, tinZaparin, tirofiban, tranexamic acid, urokinase, plicamycin, risedronate, Sodium fluoride, teriparatide, tiludr warfarin. onate, Zoledronic acid. 0267. Hypothalamic and pituitary hormones include bro 0272 Beta-lactam antibiotics and other inhibitors of cell mocriptine, cabergoline, cetrorelix, chorionic gonadotropin wall synthesis include the penicillins, such as amoxicillin, [hCG], corticorelin ovine, corticotropin, cosyntropin, desmo amoxicillin/potassium clavulanate, amplicillin, amplicillin/ pressin, follitropin alfa, follitropen beta [FSH), ganirelix, Sulbactam Sodium, carbenicillin, dicloxacillin, mezlocillin, gonadorelin acetate GnRH, gonadorelin hydrochloride nafcillin, oxacillin, penicillin G benzathine, penicillin G GnRH, goserelin acetate, histrelin, leuprolide, menotropins procaine, penicillin V, piperacillin, pipercillin and taZobac hMG, nafarelin, octreotide, oxytocin, pergolide, protirelin, tam Sodium, ticarcillin, and ticarcillin/clavulanate potassium; sermorelin, Somatrem, Somatropin, thyrotropin alpha, trip the cephalosporins and other beta-lactam drugs, such as the torelin, urofollitropin, vasopressin. narrow spectrum (first generation) cephalosporins, e.g., 0268 Thyroid and antithyroid drugs include the thyroid cefadroxil, cefaZolin, cephalexin, cephalothin, cephapirin, agents: levothyroxine T4, liothyronine T3, liotrix a 4:1 and cephradine; the second generation (intermediate spec ratio ofT4:T3, thyroid desiccated (USP); and the antithyroid trum) cephalosporins, e.g., cefaclor, cefamandole, cefineta agents: diatrizoate Sodium, iodide, iopanoic acid, ipodate Zole, cefonicid, cefotetan, cefoxitin, cefprozil, cefuroxime, Sodium, methimazole, potassium iodide, propylthiouracil andloracarbef; the broad spectrum (third- and fourth-genera IPTU, thyrotropin; recombinant human TSH. tion cephalosporins, e.g., cefdinir, cefditoren, cefepime, 0269 Adrenocorticosteroids and adrenocortical antago cefixime, cefoperazone, cefotaxime, cefpodoxime proxetil, nists include the glucocorticoids for oral and parenteral use: ceftazidime, ceftibuten, ceftizoxime, and ceftriaxone. Further betamethasone, betamethasone sodium phosphate, cortisone, classes include the carbapenem and monobactam, e.g., aztre dexamethasone, dexamethasone acetate, dexamethasone onam, ertapenem, imipenem/cilastatin, and meropenem; and Sodium phosphate, hydrocortisone cortisol, hydrocortisone other drugs such as cycloserine (seromycin pulvules), fosfo acetate, hydrocortisone cypionate, hydrocortisone sodium mycin, Vancomycin. phosphate, hydrocortisone sodium Succinate, methylpred 0273 Other antibiotics include chloramphenicol, the tet nisolone, methylprednisolone acetate, methylprednisolone racyclines, e.g., demeclocycline, doxycycline, methacycline, sodium Succinate, prednisolone, prednisolone acetate, pred minocycline, oxtetracycline, and tetracycline; the mac nisolone sodium phosphate, prednisolone tebutate, pred rolides, e.g., azithromycin, clarithromycin, erythromycin:the ketolides, e.g., tellithromycin; the lincomycins, e.g., clinda nisone, triamcinolone, triamcinolone acetonide, triamcino mycin; the streptogramins, e.g., quinupristin and dalfopristin; lone diacetate, triamcinolone hexacetonide. Another class of and the oxazolidones, e.g., lineZolid. adrenocorticoids are the mineralocorticoids, e.g., fludrocor 0274 Aminoglycosides and spectinomycin antibiotics tisone acetate. The adrenal steroid antagonists include ami include amikacin, gentamicin, kanamycin, neomycin, noglutethimide, ketoconaZole, mitotane. netilmicin, paromomycin, spectinomycin, streptomycin, and 0270 Gonadal hormones and inhibitors include the estro tobramycin. gens: conjugated estrogens, dienestrol, diethylstilbestrol diphosphate, esterified estrogens, estradiol cypionate in oil, (0275) Sulfonamides, trimethoprim, and quinolone antibi estradiol, estradiol transdermal, estradiol Valerate in oil, otics include the general-purpose Sulfonamides, e.g., sulfadi estrone aqueous suspension, estropipate, ethinyl estradiol: azine, sulfamethizole, sulfamethoxaZole, sulfanilamide, and the progestins: hydroxyprogesterone caproate, levonorg Sulfisoxazole, the Sulfonamides for Special applications, e.g., estrel, medroxyprogesterone acetate, megestrol acetate, nore mafenide, silver Sulfadiazine, Sulfacetamide sodium. Trime thindrone acetate, norgestrel, progesterone; the androgens thoprims include trimethoprim, trimethoprim-sulfamethoX and the anabolic Steroids: methyltestosterone, nandrolone azole [co-trimoxazole, TMP-SMZ]; the quinolones and fluo decanoate, Oxandrolone, oxymetholone, Stanozolol, testolac roquinolones include cinoxacin, ciprofloxacin, enoxacin, tone, testosterone aqueous, testosterome cypionate in oil, tes gatifloXacin, levofloXacin, lomefloXacin, moXifloXacin, nali tosterone enanthate in oil, testosterone propionate in oil, tes diXic acid, norfloxacin, ofloxacin, sparfloxacin, and trova tosterone transdermal system, testosterone pellets. Drugs floxacin. may further be classed as antagonists and inhibitors of 0276 Antimycobacterial drugs include drugs used in gonadal hormones: anastroZole, bicalutamide, clomiphene, tuberculosis, e.g., aminosalicylate sodium, capreomycin, danazol, dutasteride, exemestane, finasteride, flutamide, full cycloserine, ethambutol, ethionamide, isoniazid, pyraZina vestrant, letroZole mifepristone, nilutamide, raloxifene, mide, rifabutin, rifampin, rifapentine, and Streptomycin; and tamoxifen, and toremifene. drugs used in leprosy, e.g., clofazimine, dapsone. 0271 Agents that affect bone mineral homeostasis include [0277] Antifungal agents include amphotericin B, buta Vitamin E, its metabolites and analogs: calcifediol, calcitriol, conazole, butenafine, caspofungin, clotrimaZole, econazole, cholecalciferol [D3], dihydrotachysterol [DHT, doxercalcif fluconazole, flucytosine, griseofulvin, itraconaZole, keto erol, ergocalciferol [D2), and paricalcitol; calcium: calcium conazole, miconaZole, naftifìne, natamycin, nyStatin, OXi acetate 25% calcium, calcium carbonate 40% calcium, conazole, sulconazole, terbinafine, terconazole, tioconazole, calcium chloride 27% calcium, calcium citrate 21% cal tolnaftate, and Voriconazole. cium], calcium glubionate [6.5% calcium]; calcium glu 0278 Antiviral agents include abacavir, acyclovir, ade ceptate 8% calcium, calcium gluconate 9% calcium, cal fovir, amantadine, amprenavir, cidofovir, delavirdine, cium lactate 13% calcium, and tricalcium phosphate 39% didanosine, efavirenz, enfuvirtide, famciclovir, formivirsen, calcium; phosphate and phosphate binders such as phos foscarnet, ganciclovir, idoxuridine, imiquimod, indinavir, phate and sevelamer; and other drugs such as alendronate, interferon alfa-2a, interferon alpha-2b, interferon-2b, inter US 2010/013.6614 A1 Jun. 3, 2010 33 feron alfa-n3, interferon alfacon-1, lamivudine, lopinavir/ ritonavir, nelfinavir, nevirapine, oseltamivir, paliviZumab, TABLE 10 peginterferon alfa-2a, peginterferon alfa-2b, penciclovir, rib avirin, rimantadine, ritonavir, saquinavir, stavudine, teno Structural Classes of Antibiotic Drugs fovir, trifluridine, valacyclovir, Valgancyclovir, Zalcitabine, Structure Class Examples of Antibiotics within Structure Class Zanamivir, and Zidovudine. Amino Acid Derivatives AZaserine, Bestatin, Cycloserine, 0279. Further antimicrobial agents, disinfectants, antisep 6-diazo-5-oxo-L-norleucine Aminoglycosides Armastatin, Amikacin, Gentamicin, tics, and sterilants include the miscellaneous antimicrobial Hygromicin, Kanamycin, Streptomycin agents, e.g., methenamine hippurate, methenamine mande Benzochinoides Herbimycin Carbapenems Imipenem, Meropenem late, metronidazole, mupirocin, nitrofurantoin, polymyxin B; Coumarin-glycosides Novobiocin and the disinfectants, antiseptics, and sterilants, e.g., benza Fatty Acid Derivatives Cerulenin lkonium, benzoyl peroxide, chlorhexidine gluconate, glut Glucosamines 1-deoxynoirimycin Glycopeptides Bleomycin, Vancomycin araldehyde, hexachlorophene, iodine aqueous, iodine tinc midaZoles MetroidaZole ture, nitrofuraZone, oxychlorosene Sodium, providone Penicillins Benzylpenicillin, Benzathine penicillin, Amoxycillin, Piperacillin iodine, sliver nitrate, and thimerosal. Macrolides Amphotericin B, AZithromycin, Erythromycin 0280 Antiprotozoal drugs include albendazole, atova Nucleosides Cordycepin, Formycin A, Tubercidin quone, atovaquone-proguanil, chloroquine, clindamycin, Peptides Cyclosporin A, Echinomycin, Gramicidin Peptidyl Nucleosides Blasticidine, Nikkomycin doxycycline, dehydroemetine, eflornithine, halofantrine, Phenicoles Chloramphenicol, Thiamphenicol iodoquinol, mefloquine, melarsoprol, metronidazole, nifur Polyethers LasallocidA, Salinomycin Quinolones 8-quinolinol, Cinoxacin, Ofloxacin timoX, nitazoxanide, paromomycin, pentamidine, pri Steroids Fusidic Acid maquine, pyrimethamine, quinidine gluconate, quinine, Sulphonamides Sulfamethazine, Sulfadiazine, Trimethoprim Sodium Stibogluconate, Sulfadoxine and pyrimethamine, and Tetracyclins Oxytetracyclin, Minocycline, Duramycin Suramin. 0281 Anthelmintic drugs include albendazole, bithionol, 0285. In some embodiments, drugs are classed as optical diethylcarbamazine, ivermectin, levamisole, mebendaZole, isomers, where a class is two or more optical isomers, or metrifonate, niclosamide, oxamniquine, oxantel pamoate, racemate, of a compound of the same chemical formula. piperaZine, praZiquantel, pyrantel pamoate, Suramin, thia Thus, the invention includes methods and compositions for bendazole. screening individuals for a genetic variation and/or pheno 0282 Immunopharmacological agents include abcix typic variation that predicts responsiveness to a first drug, and imab, adalimumab, alefacept, alemtuZumab, anti-thymocyte using this association to determine whether or not to modu globulin, azathioprine, basiliximab, BCG, cyclophospha late the treatment of an individual with a second drug, where mide, cyclosporine, daclizumab, etanercept, gemtuZumab, the first and second drugs are optical isomers. In some glatiramer, ibritumomab tiuxetan, immune globulin intrave embodiments, the first drug is a racemate and the second drug nous, infliximab, interferon alfa-2a, interferon alfa 2b, inter is a stereoisomer that is a component of the racemate. In some feron beta-1a, interferon beta-1b, interferon gamma-1b, inter embodiments the first drug is a stereoisomer and the second leukin-2, IL-2, aldesleukin, leflunomide, levamisole, drug is a racemate that includes the Stereoisomer. In some lymphocyte immune globulin, methylprednisolone sodium embodiments the first drug is a first stereoisomer and the succinate, muromonab-CD3 (OKT3), mycophenolate second drug is a second stereoisomer of a compound. mofetil, pegademase bovine, peginterferon alfa-2a, peginter 0286. In some embodiments, drugs are classed as different feron alfa-2b, prednisone, RHo(D) immune globulin micro crystal structures of the same formula. Thus, the invention includes methods and compositions for Screening individuals dose, rituximab, sirolimus, tacrolimus [FK506], thalidomide, for a genetic variation and/or phenotypic variation that pre and trastuzumab. dicts responsiveness to a first drug, and using this association 0283 Heavy metal chelators include deferoxamine, to determine whether or not to modulate the treatment of an dimercaprol, edetate calcium calcium EDTA), penicil individual with a second drug, where the first and second lamine, Succimer, and unithiol. drugs are members of a class of drugs of the same chemical formula but different crystal structures. Structural Classes of Drugs 0287. In some embodiments, drugs are classed by struc tural components common to the members of the class. Thus, 0284. In another example of drug classification embodi the invention includes methods and compositions for screen ments, a drug may be classified according to its structural ing individuals for a genetic variation and/or phenotypic class or family; certain drugs may fall into more than one variation that predicts responsiveness to a first drug, and using structural class or family. Thus, in some embodiments, drugs this association to determine whether or not to modulate the are classified according to structure. Drugs that have a com treatment of an individual with a second drug, where the first mon action may have different structures, and often one of the and second drugs are members of a class of drugs that contain best predictors of a drugs likely action is its structure. By way the same structural component. By way of example only, a of example only, certain classes of drugs may be further drug may be structurally classified as an acyclic ureide; acy organized by chemical structure classes presented herein. lureide; aldehyde; amino acid analog; aminoalkyl ether One non-limiting example is antibiotics. Table 9, below, pre (clemastine, doxylamine); aminoglycoside; anthracycline; sents non-limiting examples of antibiotics further classified azalide; azole; barbituate; benzodiazapene; carbamate (e.g., by illustrative chemical structure classes. felbamate, meprobamate, emylcamate, phenprobamate); car US 2010/013.6614 A1 Jun. 3, 2010 34 bapenam; carbohydrate; carboxamide (e.g., carbamazepine, oligonucleotides were mixed together in equal amounts to oXcarbazepine); carotenoid (e.g., lutein, Zeaxanthin); cepha form the Y-DNA. In both cases, high-resolution gel electro losporin; cryptophycin; cyclodextrin; diphenylpropylamine; phoresis was applied to evaluate, the formation of Y-DNA expanded porphyrin (e.g., rubyrins, Sapphyrins); fatty acid; (FIG. 2). glycopeptide; higher alcohol; hydantoins (e.g., phenyloin): 0292 Nucleic acid samples were evaluated on either 3% hydroxylated anthroquinone; lincosamide; lipid, lipid related agarose ready gel (Bio-Rad, Hercules, Calif.) or 4-20% TBE compound; macrollide; mustard; nitrofuran; nitroimidazole; polyacrylaraide ready gel (Bio-Rad) at 100 volts. In some non-natural nucleotide; non-natural nucleoside, oligonucle cases, in order to confirm a particular structure, single otide; organometallic compound; oxazolidinedione; penicil stranded DNA were obtained by denaturing where NaOH was lin; phenothiazine derivative (alimemazine, promethazine); added to the nucleic acid samples to a final concentration of phenylpiperidine, phthalocyanine; piperazine derivative 20 mM. The alkalized samples were then incubated at 95°C. (e.g., cetrizine, meclozine); platinum complex (e.g., cis-pl for 2 min and cooled immediately on ice before electrophore atin); polyene; polyketide; polypeptide; porphyrin; prostag S1S. landin (e.g., misoprostol, enprostil); purine: pyraZolone; pyri 0293. The electrophoretic mobility of an oligonucleotide midine; pyrrolidine (levetiracetam); quinolone; quinone; depends on its size, shape and extent of base pairing (Kalten retinoid (e.g., isotretinoin, tretinoin); Salicylate: Sphin bach et al., J Bliomol Struct Dyn 1, 159-68 (1983), which is golipid; steroid (e.g., prednisone, triamcinolone, hydrocorti hereby incorporated by reference in its entirety). Lanes 1-3 of sone); substituted alkylamine (e.g., talastine, chlorphe FIG. 2 show the individual single DNA strands (30-mers); namine); substituted ethylene diamine (mepyramine, Y.Y., andY. Lanes 4-6 of FIG.2 represent three possible thonzylamine); Succinimide (ethoSuximide, phensuximide, combinations of Y, Y, and Y, i.e. Yoa with Yob, Y, with mesuximide); sulfa; Sulfonamide (sulfathiaZole, mafenide); Y, and Y, with Y. One major band appears on the gel. Sulfone; taXane; tetracycline (e.g., chlortetracycline, OXytet and the mobility is less than the single DNA strands, indicat racline); texaphyrin (e.g., Xcytrin, Antrin); thiazide; thiazo ing that one arm of Y-DNA has been formed. Lanes 7-9 of lidinedione; tocopherol, tocotrienol, triazine (e.g., lamot FIG. 2 show the stepwise equal molar mixtures of all three rigine); urea; Xanthine (theobromine, aminophylline); and strands, and lane 10 of FIG. 2 shows the all-in-one equal Zwitterion. molar mixtures of all three strands. There is no difference in results between stepwise and all-in-one synthesis. Dominant EXAMPLES bands, where the mobility has been retarded, suggest that Example 1 Y-DNA was formed as predicted (See FIG. 1A). The esti mated yield of Y-DNA is more than 90%. Other Y-DNA, Y, 0288 The sequences of the strands, shown in Table 1, were Y.Y., and Y, were similarly built. designed according to the standards set by Seeman (Seeman, J Biomol Struct Dyn 8:573-81 (1990), which is hereby incor Example 2 porated by reference in its entirety) and commercially syn thesized (Integrated DNA Technologies, Coralville, Iowa). Design, Construction, and Evaluation of Dendrimer All oligonucleotides were dissolved in annealing buffer (1 Like DNA using Y-DNA OmM Tris, pHS. 0.50 mM NaCl, 1 mM EDTA) with a final 0294 For constructing DL-DNA, individual Y-DNAS concentration of 0.1 m.M. were ligated specifically to other Y-DNAs, without self-liga 0289 Y-shaped DNA (Y-DNA) were synthesized by mix tion. The ligations were performed with Fast-Link DNA ing equal amounts of three oligonucleotide Strands. (see FIG. Ligase (Epicentre Technologies, Madison, Mi.). T4 DNA 1A: also as described above, supra, Y-Shape). All the mixtures ligase may also be used (Promega Corporation, Madison, were first incubated at 95°C. for 2 mM, then quickly cooled Wis.). The reaction scheme is shown in FIGS. 1B and 1C. The to 60° C., and finally slowly cooled to 4°C. nomenclature of DL-DNA is as follows: the core of the den 0290 Annealing Program: drimer, Y, is designated as G, the 0 generation of DL-DNA. After Y is ligated with Y, the dendrimer is termed the 1st generation of DL-DNA (G), and so on. Then" generation of DL-DNA is noted as G. Control block 0295. As noted above, each Y-DNA is composed of three Lid 1050 C. single DNA strands (Table 4). These strands are designed so (Denaturation) 95° C. 2 min (Cooling) 65° C. 2 min that ligations between Y, and Y can only occur when i? (no (Annealing) 60° C. 5 min self-ligation). In addition, the ligation can only occur in one (Annealing) 60° C. 0.5 min direction, that is, Y->Y->Y->Y->Y. In other words, Temperature increment -1° C. when Y is ligated to Y with 1:3 stoichiometry, threeY units (number of cycle) go to (5) Rep 40 are linked with one Y forming 1 generation of DL-DNA (Hold) 4° C. efter (G). G can then be ligated to sixY units due to the fact that there are 6 arms of Yi now (eachY posses two arms), and the 0291. In one embodiment, Y-DNAs are the basic building resulting product is a second-generation DL-DNM (G). A blocks for the DL-DNMs. Two strategies were adopted to third (G), fourth (G), and even higher generation synthesize the Y-DNA: stepwise and all-in-one. In the step DL-DNMs could be synthesized in a similar way. wise approach (FIG. 1A), two oligonucleotides with comple 0296. The first generation DL-DNM was built by ligating mentary regions formed one arm of a Y-DNA; then a third Yo and Y with 1:3 stoichiometry. The ligation product oligonucleotide, that was complementary to the first two un migrates as a single band, and its mobility is slower than that matched regions of oligonucleotides, formed the other two of its building block, Y. The presence of a single band indi arms of the Y-DNA. In die all-in-one approach, all three cates that a new molecular species with a well-defined sto US 2010/013.6614 A1 Jun. 3, 2010

ichiometry has formed. The estimated yield is more than centrate the activated oligonucleotides with either Microcon 95%.To further evaluate die structure of the ligation product, Y-3 (Bedford, Mass.) or freeze-drying. it was denatured and examined by gel electrophoresis. There (0301 Slowly mix the above SMCC-activated DNA with are two major bands for the denatured sample one with the an &fold molar excess of NLS peptide (other peptides would same mobility as the single strand DNAY (30-mer) and one be using similar; also see Note 3 for difficult peptides). Adjust with slower mobility (see arrow, a single stranded 90-mer the reaction mixture with 10xPBS so that the final solution Strand), which is exactly what one would expect according to contains 1xPBS. Incubate the reaction at room temperature the assembly scheme. Notice that denaturing G DL-DNM with gentlestining overnight. The crude product can be stored results in two sizes of single strands left: one 30-mer (Y) at — 20 OC for later processing. and the other 90-mer ((Y)(Y)(Y)(Y)(Y)(Y), and 0302 Highly cationic peptides can interact with nega (Y) (Y)(Y)). Taken together, these results indicate that tively charged oligonucleotides before a conjugation com the formation of the 1* generation of DL-DL-DNM is as pletes. This interaction can be prevented in a reaction buffer expected with high yield. with a high concentration of salt with or without organic 0297. The second, third, and fourth generation DL-DNM solvent (Vives et al. 1997, Tetrahedron Letters, 38:1183-86; were synthesized with, the stepwise approach and evaluated Astriab-Fisher et al. 2002: Pharm. Res. 19:744-54). For by gel electrophoresis. With each increased generation, the example, both SMCC functionalized oligonucleotide solu mobility of the ligated product decreased as predicted. The tion and highly cationic peptide solution are adjusted with 0.5 yield and the purity of higher generations (G and G) DL MKB2P04 (pH 7.S), 4 MKBrand urea to a final concentra DNM did not decrease, even without purification, indicating, tion of 0.1 M KH2P04 (pH 7.9, 0.3 M KBrand 8 M urea that the stepwise synthesis approach is very robust. (Astriab-Fisher et al. 2002). Acetonitrile (4096, VN) and 0.4 M KCl (Vives et al. 1997) can also be used to facilitate this Example 3 difficult conjugation. For example, adjusted peptide Solution Atomic Force Imaging of DL-DNA is slowly added to SMCC functionalized oligonucleotide Solution with stirring. The reaction mixture is then gently 0298. A 5ul DNA sample was placed onto the surface of stirred at room temperature for overnight. In addition, highly freshly cleaved mica (Ted Pella, Redding, Calif.) functional positively charged peptides can also be selectively conjugated ized with aminopropyltriethoxysilane (APTES, Aldrich) and to oligonucleotides through a disulfide bond (Vives et al. allowed to adsorb to the mica surface for approximately 20 1997: Astriab-Fisheret al. 2002) if the reduction of disilfidein minutes. The mica was then rinsed in Milli-Q water and dried the cytoplasm doesn't interfere with the downstream appli with compressed air. Images were taken in air using Tapping cations of peptideoligonucleotide. mode on a Dimensions 3100 Atomic Force Microscope 0303 Since most of the conjugation cannot reach 100% (Digital Instruments, Santa Barbarra, Calif.), and the ampli efficiency, the conjugated products need to be purified from tude setpoint was adjusted to maximize resolution while unreacted materials using 20% preparative polyacryrinide gel minimizing the force on the sample. Briefly, the amplitude electrophoresis (PAGE). The oligonucleotide-peptide conju setpoint was increased until the tip disengaged the Surface, gates are separated by conventional PAGE. Cut out the gel and then decreased by 0.1 to 0.2 volts such that the tip was slices containing the conjugated products under UV illumi engaged and applying the minimal force onto the sample nation. The gel slices are then crashed with a small Syringe Surface. Images were processed with a flattening filter. and further with shear stress created by vigorous stirring in 0299. As noted by high-resolution agarose gel electro TE buffer (10 mMTris-Cl, pH=8.0, 1 mM EDTA). Concen phoresis, different generations of dendrimers were assembled trate the purified and extracted conjugates with a Microcon from basic Y-DNA building blocks. To confirm that the gel Y-. Once sequences have been designed, evaluated and char shifted species were indeed DL-DNA molecules, the 4th gen acterized, DL-DNA can be synthesized by sequential liga eration DL-DNM was examined by AFM. FIG.5 shows clus tions ofY shaped DNA (Y-DNA) via complementary sticky ters of nanoparticles with highly branched DL-DNA ends. Note that each sticky-end is designed to be non-palin molecules. The width of DNA strands was measured to be dromic and unique so that selfligation can be totally avoided. approximately 9.0 run, consistent with the radius of curvature The nomenclature of DL-DNM is as follows: the core of the of the AFM tips. The measured diameter of 4* generation of dendrima, Yo is designated as GO (the oth generation of DL DL-DNA nanostructure was 71.2+6.7 ma, which was very DNM). After Yo is ligated with YI, the dendrima is termed the close to the theoretically calculated value (69.0 nm) consid generation 1 DL-DNM (G), and so on. Then" generation of ering the relative flexibility of DNA molecules. DL-DNM is noted as G. Example 4 Example 5 Activation and Conjugation of DL-DNM DL-DNA Assembly: Y-DNA 0300 Dissolve SMCC in organic solvent, such as dimeth ylformarnidc (DMF). Re-suspend theamino-modified oligo 0304 Each Y-DNA unit is synthesized by annealing 3 nucleotides in phosphate buffered saline (PBS, pH 7.3-7.5). single-stranded DNA with a one-pot approach. Dissolve each Mix the DNA with a 40:1 molar excess of SMCC in DMF. oligonucleotide strand in an annealing buffer (10 rnM Tris pH Incubate the reaction mixture in the dark at room temperature 8.0, 1 mM EDTA). Combine each oligonucleotide in an equal for 2 hours. Remove free SMCC from activated protein, pep molar ratio in a microcentrifuge tube. Increase temperature to tide or oligonucleotide through filtration, for example via 95° C. for 5 minutes to denature all oligos. Anneal oligos at Sephadex TM G-25, by simple centrifugation. The excess 65° C. for 2 minutes. Anneal oligos at 62° C. for 1 minute. SMCC can also be removed with a desalting column (Bio Then linearly decrease temperature at a rate of 2°C./minute Rad, Hercules, Calif.) using water as the elution buffer. Con for 20 minutes. Y-DNA will be formed. If this Y-DNA is used US 2010/013.6614 A1 Jun. 3, 2010 36 as a core to grow further generation DL-DNM, then this Further cooling at 25°C. for 1 hr with a continuous tempera Y-DNA is also called G-DNA. Store Y-DNA at 4°C. ture decrease at a rate of 0.50 C per min. The final annealed 0305 DL-DNA Assembly: Generation I products are stored at 40C. (0306 Combine G and 3 Y-DNA in the appropriate molar 0314 DL-DNA Solid Phase Assembly: Generation 1 ratio (1:3). Add 10% volume of T4 Ligase buffer (5ul for 50 0315] Place 100 ul ofavidin coated agarose beads in a 1 ml ul reaction volume) and mix well. Add T4 Ligase based on the microcentrifuge tube and then add 1.3 ml of SDS solution to enzymatic activity specified on the T4 tube.G will beformed pre-treat the avidin beads. The solution is mixed at 15 rpm after ligation at room temperature for 16 hours. rotation for 15-30 min. (Step I in Scheme 0). Add 150 ul(8.2 0307 DL-DNA Assembly: Generation 2 and Beyond nmole) of spacer DNA into the microcentrifuge tube and then 0308 Combine G and 6Y-DNA in the appropriate molar react in a rotary incubator overnight at room temperature. ratio (1:6) to form G. Repeat ligation steps listed above for (Step I in Scheme I). The resulting avidin coated beads con G synthesis. Repeat this procedure to generate higher gen taining spacer DNA are centrifuged at 2.5 kG and washed eration DL-DNM. G3+12Y-DNA- >Ga; G+24Y-DNA->Ga; with sterile Milli-Q water. (Step I in Scheme I). To grow G+48 Y-DNA->Gs. DL-DNM on beads, individual Y-DNA is ligated specifically to a spacer or otherY-DNA. For example, G DL-DNM can be Example 6 obtained by ligating Y to the spacer-modified bead. (Step II in Scheme I). Similarly, G is formed by ligating two Y with DL-DNM Synthesis: Solid Phase Approach one G. (Step III in Scheme I). Other higher generations of DL-DNM are constructed using the same strategy. Each liga 0309. A solid phase approach provides a more robust syn tion reaction solution contains 8.0 nmole of Y-DNA, 2.1 thetic route that combines assembly and purification in one Weiss unit of T4 DNA ligase, ligasebuffer (300mM Tris-HCl step. The products are more pure, and the overall yield is in pH 7.8, 100 mM MgCl2, 100 mM DTT) and 10 mM ATP. much higher than Solution-based synthesis. An extra spacer (Step 1 V-VI in Scheme I). After ligation, the DL-DNM is DNA is needed to attach Y-DNA or DL-DNM onto a solid cleaved off from the solid phase by the restriction enzyme, Surface. Sample sequences are listed in Table 4A and 4B, and DDE I. The enzyme solution contained 10 ul of a DDE I and sample spacer sequences herein below. The scheme of Solid Bovine Serum Albumin (BSA) and restriction buffer D with phase synthesis of DL-DNA is depicted in FIGURE XXX. 60 mM Tris-HCl in pH 7.9, 1.5 M NaCl, 60 mM MgCl2 and 10 mM DTT. (Step VII in Scheme I). Spacer 1 Biotin-5'-p 0316 Testing DNA Delivery using the DL-NAMs System (SIEQ IID INO :: 76 ) 0317. Once DL-NAM is synthesized and functionalized CCGGATAAGGCGCAGCGGTCGGCTGAATTCAGGGTTCGTGGCAGGCCAGC Subsequently with multi-functional components, it can be used directly in delivering genes and other nucleic acids ACACTTGGACGACCCGAAGCTTACCGCGACTCCTA AC-3 " (RNAi, for example). The procedures for evaluating cytotox Spacer 2 5 " — p — TCA icity and delivery efficiency have been outlined herein and are (SIEQ IID INO :: 77 ) known in the art. It is important to note that the DL-NAMs GTTAGGAGTCOGGTAAGOTTOGGTCTOCAAGT GTGOTGGCCTGCCACGA system is a dynamic system in that it is totally modular by ACCCTGAATTCAGCCGACCGCTGCGCCTTATCCGG-3 ! design; thus, one can easily “mix and match' different com ponents and “plug and play” to test delivery behaviors. This 0310 DL-DNA Solid Phase Assembly: Y-DNA DL-NAM system provides a platform technology to conju 0311. Without further purification, oligonucleotides, Y, gate a variety of receptors and other targeting molecules, Y., andY., are dissolved in annealing buffer (10 mM Tris, 1 making targeted delivery possible. Our results indicate that mMEDTA, 50 mM NaCl, pH 8.0) with a final concentration cytotoxicity is very low with the DL-NAM systeni. of 0.2 mM. To construct Y-DNA, three oligonucleotide com 0318. A major advantage of this system is the built-in ponents, Y, Y, and Y (1:1:1 molar ratio) are mixed in modularity resulting in great flexibility. Both viral and non sterile Milli-Qwater with a final concentration of 40 uM for viral components can be attached specifically. Pre-made each oligonucleotide. Hybridizations are performed accord modules will further increase flexibility and make “plug-and ing to the following procedures: (i) Denaturation at 95°C. for play' possible. Such flexibility is especially useful in study 2 min; (ii) Cooling at 65° C. and incubation for 2 min; (iii) ing the complex processes of DNA delivery because, one, Annealing at 60 0 C for 5 mM; and (iv) Further annealing at little is known quantitatively about intracellular events, and 60° C. for 0.5 min with a continuous temperature decrease at two, one can easily adjust the delivery vector based on the a rate of 1° C. per min. The annealing steps were repeated a experimental outcomes. In addition, the DL-NAM system is total of 40 times. The final annealed products were stored at 4 capable of carrying both genes and anti-genes (siRNA), as C well as other entities such as enzymes and chemical drugs. A 0312 DL-DNA Solid Phase Assembly: Spacer DNA combination of DNA vaccination, gene therapy, antibody/ 0313 Two oligonucleotides are synthesized commer enzyme therapy and si-RNA therapy is thus possible. cially: SP1 and SP2: one of them (SP1) is 5'-biotin modified. Examples for modes of delivery of nucleic acids, including Each oligonucleotide is dissolved in a 1xPBS buffer (10 mM for vaccination, are known in the art, as disclosed in U.S. Pat. phosphate; pH=7.4, 2.7 mMKC1, 137 mMNaCl) with a final No. 6,946,448; 6,893,664; 6,821,955; 6,689,757 or 6,562, concentration of 0.2 mM. The spacer is assembled by hybrid 8O1. izing two oligonucleotide components (1:1 molar ratio) in 0319. Furthermore, the size of a DL-NAM vector is sterile Milli-Qwater with a final concentration of 60 uM for designed and constructed at the nanoscale, which is important each oligonucleotide. Hybridizations are performed accord in intracellular DNA delivery as well as cellular targeting. For ing to the following procedures: (i) Denaturation at 94°C. for example, in one embodiment of the invention, the capability 4 min; (ii) Annealing at 80° C. and incubation for 2 min; (iii) of adding multiple modules, DL-DNA-based DL-NAM pro US 2010/013.6614 A1 Jun. 3, 2010 37 vides an advanced platform for constructing an artificial change combinations of different targeting moieties or virus' that utilizes useful viral components to mimic multiple change targeting moieties altogether. viral functions for DNA delivery with no fear of any viral infection. This DL-DNA-based, nanoscale DL-NAM will Example 9 play an important role in biomedical and pharmaceutical research. Gene Delivery Example 7 0324. This example provides results that illustrate that a DL-NAM can be utilized to overcome three currently identi 0320 Construction of the DNA building block X-DNA. fied gene delivery barriers at the cellular level: (1) DNA The branched DNA sequences (Table 1) were designed and condensation and protection; (2) crossing the plasma mem synthesized using commercially available oliognucleotide brane via transduction without endocytosis; and (3) nuclear synthesis. Without further purification, oligonucleotides (In targeting and entry. tegrated DNA Technologies, Coralville, Iowa) were dis 0325 Viral peptides were utilized, thus essentially, pro solved in an annealing buffer (10 mM Tris, pH=8.0, 1 mM ducing a hybrid viral/non-viral vector (i.e., DL-NAM with ethylenediaminetetraacetic acid (EDTA), and 50 mM NaCl) viraltargeting peptides). For example, Adenou (mu) peptide, with a final concentration of 50 mM. X-DNA was constructed HIV-1 Rev-NLS or HIV-1 Tat were linked to DL-NAMs. All by mixing four oligonucleotide components (with the same viral peptides were synthesized with an extra amino acid, molar ratio) insterile Milli-Q water with a final concentration Cys, attached at their C-termina to introduce a free thiol of 20 mM for each oligonucleotide. Hybridizations were per group. In addition, an amine was added to the 5' end of the formed according to the following procedures: (i) denatur DNA with amino modifier C6 to introduce a primaryamine ation at 95°C. for 2 min. (ii) cooling at 65° C. and incubation group. Conjugation of viral peptides to DL-NAMs was for 2 min. (iii) annealing at 60° C. for 5 min. and (iv) further achieved through heterobifunctional crosslinkers such as annealing at 60°C. for 0.5 min with a continuous temperature SMCC that crosslinks between amine and thiol groups. Since decrease at a rate of 1° C. per min. The annealing steps were each component was designed with only one reactive amine repeated a total of 40 times. The final annealed products were or thiol group, the reactive group was monovalent, and the stored at 4°C. The Xo to X were four corresponding single reaction was terminal. Single stranded DNA was first conju oligonucleotides that formed an X-DNA. gated with a viral peptide before self-assembled into a viral Y-DNA FIG. 19. Conjugations of each viral peptide to single Example 8 stranded DNA were all successful. Functions of each viral Y-DNA was tested and confirmed either separately or com Screening Assay binatorially. For example, DNA condensation via Tat-Y-DNA was evident on gel electrophoretic retardation assay, which 0321. This example describes the binding to cells of to a showed that Tat-Y-DNA had higher mobility as compared to DL-NAM vector comprising a targeting protein (e.g., for condensed DNA without Tat. illustrative purposes only, Such as a chimeric fiber protein) as 0326. A plasmid DNA (pVax/Lacz) coding for the Lacz compared a control DL-NAM not containing a targeting moe gene was used as a reporter gene. When tagged with fluores ity, either in the presence or absence of added soluble said cent dye FAM, the fate of the hybrid vectors was followed in targeting protein. real time. Lac Z gene expression was quantified and compared 0322 For these experiments, cells can be selected based with various lipid-based vectors. The results showed that the on expection to bind with either high efficiency (i.e. receptor DL-NAMs successfully crossed the cell plasma membrane plus cells) or low efficiency (i.e., receptor-minus cells). For by viral peptides. In addition, gene expression utilizing DL example, an epithelial cell line A549 can be used as represen NAMs was substantially higher as compared to controls. tative of receptor-plus cells, and the fibroblast cell line HS 68 0327. As noted above, a major advantage of this hybrid can be used as representative of receptor-minus cells. Con system is the built-in modularity. Both viral and non-viral fluent monolayers of either A549 or HS 68 cells can be pre components can be attached specifically and independently. incubated at 4.degree. C. with concentrations of soluble fiber Preformed modules will further increase the flexibility and protein ranging from 0 to about 10 mu.g/ml. The DL-NAM make “plug-n-play a reality using DL-NAMs as a platform. vector comprising the targeting moeity (T+) or control vector Moreover, DL-NAM conjugation (e.g., by varying concen comprising no fiber protein (WT) can be labeled with tritiated trations of DL-NAM, or polynucleotides to target) can be thymidine. About 20,000 cpm of HI-thymidine labeled DL easily altered based on cell culture or animal model experi NAM or WT vector can be incubated with the cells for about ments. Key advantages of the DL-NAM are its nanoscale size 2 hours at 4°C. The cells were washed three times with cold (allowing intracellular delivery), modularity (allowing drugs, PBS, and the cell-associated cpm is determined by scintilla antibody, enzymes, nucleic acids to be incorporated in a con tion counting. Results obtained can be the average of dupli trolled fashion), and a nucleic acid backbone (which will cate measurements and are presented for the A549 and HS 68 eventually be degraded into its natural monomers, thus bio cell lines, respectively. degradable and biocompatible). 0323. Therefore, based on the readings from the scintilla 0328. Although the foregoing invention has been tion counting receptor-plus and receptor-minus cells will described in some detail by way of illustration and example indicate whether a particular particular moiety or concentra for purposes of clarity of understanding, it is readily apparent tion of a particular particular moiety, as correlated to a par to those of ordinary skill in the art in light of the teachings of ticle cell or cell receptor, is effective with high efficiency to this invention that certain changes and modifications may be effectuate cellular uptake. A DL-NAM can be modified made thereto without departing from the spirit or scope of the accordingly to increase the number of targeting moieties or to appended claims.

US 2010/013.6614 A1 Jun. 3, 2010 40

- Continued

FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

SEQUENCE: 12

SEQ IID NO 13 LENGTH: 16 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

SEQUENCE: 13 tggacgt.cta cc.gtgt 16

SEQ IID NO 14 LENGTH: 15 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

SEQUENCE: 14 acgcatac ca t ccag 15

SEQ IID NO 15 LENGTH: 6 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

SEQUENCE: 15 gCctgC

SEQ IID NO 16 LENGTH: 30 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

SEQUENCE: 16 ctgagtacct gattggctta gtcggaagct 3 O

SEQ IID NO 17 LENGTH: 30 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

SEQUENCE: 17 acct aggcgt actatcaggt actcagttaa 3 O

SEQ IID NO 18 LENGTH: 30

US 2010/013.6614 A1 Jun. 3, 2010 43

- Continued

< 210 > SEQ IID NO 3 O &211s LENGTH: 13 < 21-2 > TYPE : DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

<4 OO> SEOUENCE: 3 O gaatgccgct tac 13

< 210 > SEQ IID NO 3 1 &211s LENGTH: 13 < 21-2 > TYPE : DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

<4 OO> SEOUENCE: 31 t ccg acta ag c ca 13

< 210 > SEQ IID NO 3 2 &211s LENGTH: 30 < 21-2 > TYPE : DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

<4 OO> SEOUENCE: 32 tgactggat c cdcatgacat tcgccgtaag 3 O

< 210 > SEQ IID NO 33 &211s LENGTH: 30 < 21-2 > TYPE : DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

<4 OO> SEOUENCE: 33 gt catggat C C gCatig ac at t cg cc gta ag 3 O

< 210 > SEQ IID NO 3 4 &211s LENGTH: 30 < 21-2 > TYPE : DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

<4 OO> SEOUENCE: 3 4 atcgtggat c cdcatgacat tcgccgtaag 3 O

< 210 > SEQ IID NO 3 5 &211s LENGTH: 30 < 21-2 > TYPE : DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

<4 OO> SEOUENCE: 35 US 2010/013.6614 A1 Jun. 3, 2010 44

- Continued atgctggat c cdcatgacat tcgccgtaag 3 O

< 210 > SEQ IID NO 3 6 &211s LENGTH: 30 < 21-2 > TYPE : DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

<4 OO> SEOUENCE: 36 gCaatggat C C gCatig ac at t cg cc gta ag 3 O

< 210 > SEQ IID NO 3 7 &211s LENGTH: 30 < 21-2 > TYPE : DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

<4 oo > SEOUENCE: 37 tgaccttacg gcgaatgacc gaatcagcct 3 O

< 210 > SEQ IID NO 3 8 &211s LENGTH: 30 < 21-2 > TYPE : DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

<4 OO> SEOUENCE: 38 cgatcttacg gcgaatgacc gaatcagcct 3 O

< 210 > SEQ IID NO 39 &211s LENGTH: 30 < 21-2 > TYPE : DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

<4 OO> SEOUENCE: 39 gcatcttacg gcgaatgacc gaatcagcct 3 O

< 210 > SEQ IID NO 4 0 &211s LENGTH: 30 < 21-2 > TYPE : DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

<4 OO> SEOUENCE: 4 O ttgccttacg gcgaatgacc gaatcagcct 3 O

< 210 > SEQ IID NO 41 &211s LENGTH: 30 < 21-2 > TYPE : DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

US 2010/013.6614 A1 Jun. 3, 2010 49

- Continued

SEQ IID NO 65 LENGTH: 26 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

SEQUENCE: 65 tggat C CgCa tga Catt CgC Cgt aag 26

SEQ IID NO 6 6 LENGTH: 30 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

SEQUENCE: 66 gcatcttacg gcgaatgacc gaatcagcct 3 O

SEQ IID NO 6 7 LENGTH: 30 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

SEQUENCE: 67 gcaacttacg gcgaatgacc gaatcagcct 3 O

SEQ IID NO 68 LENGTH: 26 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

SEQUENCE: 68 cttacggcga atgaccgaat cagcct 26

SEQ IID NO 6 9 LENGTH: 30 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

SEQUENCE: 69 gCataggctg attcggttca toggat.cca 3 O

SEQ IID INO 7 O LENGTH: 30 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

SEQUENCE: 7 O US 2010/013.6614 A1 Jun. 3, 2010 50

- Continued ttgcaggctg atticggttca toggat.cca 3 O

SEQ IID INO 71 LENGTH: 30 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

SEQUENCE: 71 aacgaggctg atticggttca toggat.cca 3 O

SEQ IID INO 72 LENGTH: 26 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

SEQUENCE: 72 aggctgattic ggttcatgcg gatcca 26

SEQ IID INO 73 LENGTH: 4 O TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

SEQUENCE: 73 acgt.cgaccg atgaat agcg gtcagat.ccg tacct acticg 4 O

SEQ IID INO 74 LENGTH: 4 O TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

SEQUENCE: 74 acgt.cgagta ggtacggatc. tcgt attgc gaacgacticg 4 O

SEQ IID INO 75 LENGTH: 4 O TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

SEQUENCE: 75 a Cgt Cgagt C gitt CgCaata cgg ctgta Cg tatggt Ct Cg 4 O

SEQ IID INO 76 LENGTH: 4 O TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide US 2010/013.6614 A1 Jun. 3, 2010 51

- Continued SEQUENCE: 76 acgt.cgagac catacgtaca gcaccgct at t catcggtcg 4 O

SEQ IID INO 77 LENGTH: 4 O TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

SEQUENCE: 77 acgt.cgaccg atgaat agcg gtcagat.ccg tacct acticg 4 O

SEQ IID INO 73 LENGTH: 4 O TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

SEQUENCE: 78 acgt Cgagt C gtt CgCaata Cgaccgct at tcat Cggt cg 4 O

SEQ IID INO 79 LENGTH: 4 O TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

SEQUENCE: 79 acgt.cgagta ggtacggatc. tcgt attgc gaacgacticg 4 O

SEQ IID INO 80 LENGTH: 4 O TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

SEQUENCE: 8O acgtcgacag ctgact agag tcacgacctg tacctactcg 4 O

SEQ IID INO 81 LENGTH: 47 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

SEQUENCE: 81 acgtcgagtc gttctcaaga cgtagctagg actictagt ca gctgtcg 47

SEQ IID INO 82 LENGTH: 4 O TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic US 2010/013.6614 A1 Jun. 3, 2010 52

- Continued oligonucleotide

<4 OO> SEOUENCE: 82 acgt Cgagt a ggt acaggt C gt cgtcttga gaacgact Cg 4 O

< 210 > SEQ IID INO 83 &211s LENGTH: 12 < 212 > TYPE : PRT < 213 > ORGANISMI : Semian virus 4 0

<4 OO> SEOUENCE: 83 Phe Lys Lys Lys Arg Lys Val Glu Asp Pro Tyr Cys 1. 5 1O

< 210 > SEQ IID INO 84 &211s LENGTH: 13 < 212 > TYPE : PRT <213> ORGANISM: Human immunodeficiency virus

<4 OO> SEOUENCE: 84 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln 1. 5 1O

< 210 > SEQ IID INO 85 &211s LENGTH: 19 < 212 > TYPE : PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OO> SEOUENCE: 85 Met Arg Arg Alla His His Arg Arg Arg Arg Alla Ser His Arg Arg Met 1. 5 1O 15 Arg Gly Gly

< 210 > SEQ IID INO 8 6 &211s LENGTH: 84 < 21-2 > TYPE : DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OO> SEOUENCE: 86 ccggat a agg CgCagCggt C ggctgaatt C agggtt Cgtg gCaggccagC a cacttggag 6 O a ccgaagcitt a ccgga ct cc taac 84

< 210 > SEQ IID INO 87 < 211 > LENGTH : 87 < 21-2 > TYPE : DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

<4 oo > SEOUENCE: 87 t cagttagga gtc.cggtaag Ctt.cggtctic Caagttgttgct ggcctgccac galacc Ctgaa 6 O tt Cagc cg ac cgc tgcgc ct t at CC gg 87

< 210 > SEQ IID INO 88

US 2010/013.6614 A1 Jun. 3, 2010 54

- Continued oligonucleotide

SEQUENCE: 93 galait gC ggct tacagtacgC C taggittagC 3 O

SEQ IID NO 9 4 LENGTH: 30 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

SEQUENCE: 94 tctic cacgct aactctgtgt catcgtactg 3 O

SEQ IID NO 9 5 LENGTH: 30 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

SEQUENCE: 95 acgatig ac ag aga atggt Cg Caagt CCtag 3 O

SEQ IID NO 9 6 LENGTH: 29 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

SEQUENCE: 96 acttgcgacc atgttagcgt ggagat.cga 29

SEQ IID NO 9 7 LENGTH: 30 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

SEQUENCE: 97 ctgagtacct gattggctta gtcggaagct 3 O

SEQ IID NO 9 8 LENGTH: 30 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

SEQUENCE: 98 acct aggcgt actatcaggt actcagttaa 3 O

SEQ IID NO 9 9 LENGTH: 30 TYPE: DNA ORGANISM: Artificial Sequence US 2010/013.6614 A1 Jun. 3, 2010 55

- Continued

22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

<4 OO> SEOUENCE: 99 gaatgccgct tacagtacgc Ctaggitticga 3 O

< 210 > SEQ IID INO 1 00 &211s LENGTH: 30 < 21-2 > TYPE : DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

<4 OO> SEOUENCE: 100 t ccg acta ag C Cagta ag Cg gcatt Cctag 3 O

< 210 > SEQ IID INO 1 01 &211s LENGTH: 29 < 21-2 > TYPE : DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

<4 OO> SEOUENCE: 101 acttgc gacc atgttagcgt ggagat.cga 29

< 210 > SEQ IID INO 102 &211s LENGTH: 60 < 21-2 > TYPE : DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

<4 OO> SEOUENCE: 102 tctic cacgct aactctgtgt catcgtactg acgatgacac agaatggtcg caagt cittaa 6 O

< 210 > SEQ IID INO 103 &211s LENGTH: 26 < 21-2 > TYPE : DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

<4 OO> SEOUENCE: 103 gact tg Cg ac Catgitt ag Cg tggaga 26

< 210 > SEQ IID INO 104 &211s LENGTH: 60 < 21-2 > TYPE : DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

<4 OO> SEOUENCE: 104 tctic cacgct aactctgtgt catcgt.ca.gt accatgagac agaatggtcg caagt cc tag 6 O

< 210 > SEQ IID INO 105 &211s LENGTH: 4

US 2010/013.6614 A1 Jun. 3, 2010 57

- Continued <4 OO> SEOUENCE: 110 Glu Pro Arg Gly Asp Asn Tyr Arg 1. 5

< 210 > SEQ IID INO 111 &211s LENGTH: 4 < 212 > TYPE : PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OO> SEOUENCE: 111 Arg Gly Asp Ser 1.

< 210 > SEQ IID INO 112 &211s LENGTH: 5 < 212 > TYPE : PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OO> SEOUENCE: 112 Glu, Ille Lueu. Asp Val 1. 5

< 210 > SEQ IID INO 113 &211s LENGTH: 4 < 212 > TYPE : PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OO> SEOUENCE: 113 Arg Glu Asp Val 1.

< 210 > SEQ IID INO 114 &211s LENGTH: 5 < 212 > TYPE : PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OO> SEOUENCE: 114 Tyr Ile Gly Ser Arg 1. 5

< 210 > SEQ IID INO 115 &211s LENGTH: 6 < 212 > TYPE : PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OO> SEOUENCE: 115 Ser Ile Lys Val Alla Val 1. 5 US 2010/013.6614 A1 Jun. 3, 2010 58

- Continued < 210 > SEQ IID INO 116 &211s LENGTH: 4 < 212 > TYPE : PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OO> SEOUENCE: 116 Arg Gly Asp Val 1.

< 210 > SEQ IID INO 117 &211s LENGTH: 7 < 212 > TYPE : PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OO> SEOUENCE: 117 His Arg Asn Arg Lys Gly Val 1. 5

< 210 > SEQ IID INO 118 &211s LENGTH: 5 < 212 > TYPE : PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OO> SEOUENCE: 118 Llys Llys Gly His Wall 1. 5

< 210 > SEQ IID INO 119 &211s LENGTH: 25 < 212 > TYPE : PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: Wariable amino acid 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (24) . . (24) <223> OTHER INFORMATION: Wariable amino acid

<4 OO> SEOUENCE: 119 Xaa Pro Gln Pro Asn Pro Ser Pro Alla Ser Pro Val Val Val Gly Gly 1. 5 1O 15 Gly Alla Ser Leu Pro Glu Phe Xaa Tyr 2O 25

< 210 > SEQ IID INO 120 &211s LENGTH: 16 < 212 > TYPE : PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES US 2010/013.6614 A1 Jun. 3, 2010 59

- Continued <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: Wariable amino acid

<4 OO> SEOUENCE: 120 Ala Ser Pro Val Val Val Gly Gly Gly Alla Ser Leu Pro Glu Phe Xaa 1. 5 1O 15

1. A composition comprising a protein manufacturing gel 13. A method for cell-free syntheses of one or snore pro comprising a polymer matrix, said matrix having at least a teins comprising expressing one or more proteins from a portion thereof formed with a plurality of nucleic acid mol matrix comprising a plurality of branched nucleic acid mono ecules. CS. 2. A composition comprising as DNA hydrogel for protein 14. A method forcell-free synthesis of one or more proteins expression comprising a polymer matrix and a nucleic acid comprising expressing one or more proteins from a DNA molecule encoding said protein. hydrogel. 3. A composition comprising a DNA hydrogel for protein 15. Akit comprising a DNA hydrogel and macromolecules expression comprising nucleic acid molecules that form a necessary for protein expression. three-dimensional matrix. 16. A kit comprising X-shape DNA, ligase and macromol 4. A composition comprising a protein manufacturing ecules necessary for protein expression. hydrogel comprising a polymer matrix formed of a DNA 17. A method of producing a protein manufacturing gel hydrogel, wherein a nucleic acid molecule is linked to at least comprising, forming a polymer matrix, said matrix having at a portion of the polymer matrix, whereby said nucleic acid least as portion thereof formed with a plurality of nucleic acid molecule encodes a protein to be expressed. molecules. 5. A composition comprising as hydrogel. comprising a 18. The method of claim 17, wherein said matrix is com three-dimensional matrix, wherein said matrix effects expres prised of nucleic acid molecules that have X-Y-, T-, dumb sion of as protein at A level Of at least 1 mg per cm of said bell, dendrimer-shape, or a combination thereof. matrix. 19. The method of claim 17, wherein said nucleic acid 6. The composition of any of claims 1 to 4, wherein said molecules are X-shape. matrix produces said protein at a level of at least 500 ug per 1 20. The method of claim 17, wherein said nucleic acid ug of nucleic acid molecules. molecules are Y-shape. 7. The composition of any of claims 1 to 4, wherein said 21. The method of claim 17, wherein said nucleic acid matrix comprises pores. molecules comprise DNA and/or RNA. 8. The composition of any of claims 1 to 4, wherein said 22. The method of claim 17, wherein said nucleic acid matrix comprises a component selected from a group consist molecules are linked to each other covalently and/or non ing of poly (N-isopropylacrylamide), poly(N-alkylacryla covalently. mide), poly(N-n-propylacrylamide), poly(N-isopropyl 23. The method of claim 17, wherein said matrix is further methacrylamide), polyethylene oxide)-poly(propylene comprised of a plurality of peptide molecules that function as oxide)-poly(ethylene oxide), poly(DTEC), dextran-polylac structural Support. tide, elastin-like polypeptides, a polyester, polylactide, poly 24. A method of producing proteins comprising, express (L.-lactic acid), poly(D.L.-lactic acid), poly(lactide-co-gly ing one or more proteins from a hydrogel that comprises collides), biotinylated poly(ethylene glycol-block-lactic coding and non-coding nucleic acid molecules. acid), poly(alkylcyanoacrylate), poly(epsilon-caprolactone), 25. A method of producing modified proteins comprising, polyanhydride, poly(bis(p-carboxyphenoxy) propane-Seba expressing. one or more proteins from a hydrogel that com cic acid), polyorthoester, polyphosphoester, polyphosp prises nucleic acid molecules and one or more macromol haZene, polystyrene, polyurethane, poly(amino acid), and a ecules necessary for protein modification, thus producing derivative of any thereof. modified proteins. 9. The composition of any of claims 1 to 4, wherein said 26. The method of claim 25, wherein said modifications matrix comprises nucleic acid molecule that comprise DNA include phosphorylation, glycosylation, methylation, ubiq having a total concentration of about 0.005, 0.0025, 0.01, uitination, biotinylation, alkylation, acetylation, glutamyla 0.02, 0.03, 0.04, 0.05, 0.00, 0.07, 0.08, 0.09 or 0.10 mM. tion, glycylation, isoprenylation, lipoylation, phosphoanteth 10. The composition of claim 7, wherein said pores are einylation, sulfation, citrullination, deamidation, from about 50 rim to 500 nm in size. isomerization, or a combination thereof. 11. The composition of claim 7, wherein said pores have a 27. The composition of any of claims 1 to 4, wherein said size selected from a group consisting of about 5 nm, about 10 composition comprises at least one macromolecule involved nm, about 15 nm, about 20 nm, about 30 nm, about 40 nm, in protein modification. about 50 nm, and about 100 rim. 28. The composition of claim 27, wherein said at least one 12. A method for in vitro syntheses of one or more proteins, macromolecule modifies a protein by phosphorylation, gly comprising expressing one or more proteins from a protein cosylation, methylation, ubiquitination, biotinylation, alkyla manufacturing hydrogel. tion, acetylation, glutamylation, glycylation, isoprenylation, US 2010/013.6614 A1 Jun. 3, 2010 lipoylation, phosphoantetheinyiation, sulfation, citrullina 33. The compositions of any of claims 1, wherein said tion, deamidation, glycosyltransferase, glycosidase, transg composition is capable of said protein manufacturing for at lycosidase or isomerization. least about 1, 2, 3, 4, 5, 6 or 7 days. 29. The composition of claim 27, wherein said glycosyla 34. The composition of any of claims 1 to 4, wherein said tion is N- or O-glycosylation. composition further comprises at least one nucleic acid mol 30. The composition of any of claims 1 to 4, wherein said ecule cross-linked to an agent that functions to stabilize said composition comprises an extract that provides the necessary matrix. macromolecules that function in post-translational modifica 35. The composition of claim 34, wherein said agent is a tion of a protein. nanoparticle. 31. The composition of claim 30, wherein said extract is 36. The composition of claim 34, wherein said agent is from a eukaryotic or prokaryotic cell. gold. 32. The composition of claim 31, wherein said eukaryotic cell is a Chinese hamster ovary (CHO) cell.