Design and Synthesis of Dendritic Molecular Transporter that

Jun 20, 2008 - This work describes the design and synthesis of a dendritic transporter in a most concise manner where the selection of the core scaffo...
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Bioconjugate Chem. 2008, 19, 1464–1470

Design and Synthesis of Dendritic Molecular Transporter that Achieves Efficient in ViWo Delivery of Morpholino Antisense Oligo Yong-Fu Li* and Paul A. Morcos Gene Tools, LLC, One Summerton Way, Philomath, Oregon 97370. Received April 4, 2008; Revised Manuscript Received May 15, 2008

Safe and efficient in ViVo delivery of Morpholino antisense oligos was probably the last and most difficult challenge for the broad application of antisense in animal research and therapeutics. Several arginine-rich peptides effective for in ViVo delivery of Morpholino antisense oligos require rather complex and expensive procedures for synthesis and conjugation. This work describes the design and synthesis of a dendritic transporter in a most concise manner where the selection of the core scaffold, functional group multiplication, orthogonal protecting group manipulation, solid phase conjugation, and off-resin perguanidinylation of the transporter structure are all orchestrated for efficient assembly. We utilized triazine as a core to provide a site for on-column conjugation to the Morpholino oligo and to anchor functional side arms which, after extension, multiplication, and deprotection, are subsequently converted from primary amines to the eight guanidinium headgroups that serve for transport across cell membranes. Intravenous administration of the delivery-enabled Morpholino into a splice-reporter strain of transgenic living mice results in de noVo expression of splice-corrected green fluorescent protein in a broad range of tissues and organs in those treated mice. This rigorously demonstrates that this new dendritic transporter achieves effective delivery of a Morpholino oligo into the cytosol/nuclear compartment of cells systemically in ViVo. The practical conjugation process may overcome any availability limitation for routine use by the scientific community, and the efficient delivery ability of this transporter may advance the application of Morpholino antisense technology in animals.

INTRODUCTION Unlike other types of antisense oligos including siRNA, PNA, mPNA, S-DNA, and LNA, Morpholinos provide all the desired properties of stability, nuclease resistance, high efficacy, long-term activity, water solubility, low toxicity, and exquisite specificity (1, 2). However, biological barriers typically limit or preclude uptake, and therefore therapeutic benefit, of large polar structures such as antisense oligos (3). Advanced antisense oligo types faced the lack of a safe and effective delivery component which could transport oligos into the cytosol/nuclear compartment of cells of living animals, and this constituted the last major hurdle before antisense oligos could be introduced for the treatment of a broad range of currently intractable diseases such as splicemodifying genetic defects and viral diseases. The development of novel guanidinium-rich compositions that facilitate cellular uptake of a variety of physiologically and therapeutically active biomolecules started with discovery that HIVTat peptide and other arginine-rich derivatives could achieve efficient intracellular delivery (4–8). These and other cellpermeable arginine-rich oligopeptides were capable of transporting various types of cargo, such as small polar molecules, oligonucleotides, plasmid DNA, and proteins, across the membranes of most types of animal cells (9–15). Structural variations on the arginine-rich peptides have shown that the length of the backbone and the spacing and composition of the side chains can be varied to adjust the uptake efficiency (16–19). Testing structural modifications on the arginine-rich peptides and peptoids have demonstrated that the guanidinium headgroups of the arginine residues are principally responsible for the uptake into cells, backbone chirality is not critical * Corresponding author. Telephone: 541-929-7840, ext. 1035. Fax: 541-929-7841. E-mail: [email protected].

for cellular uptake, and about 7 to 15 guanidine headgroups are optimal for efficient uptake (20–22). Arginine-rich peptides have been covalently conjugated with Morpholino antisense oligos and the conjugates were shown to enter the cytosol and nucleus of cells (23–26). Because of the peptidic nature, however, degradation of the peptide portion of the conjugates was found to be time- and tissue-dependent ( (27) and references cited therein). In addition, the intensively studied cell-penetrating peptides [(R-AhxR)4AhxB] showed different degrees of toxicity depending on the dose, dose frequency, and route of administration. From a practical point of view, the application of such arginine-rich peptide transporters is somewhat limited due to their high cost, scalability, and stability. Usually, these peptide transporters are prepared using a solid-phase synthesizer. Although this approach is readily automated and allows for efficient synthesis and purification of the transporter peptides, the approach suffers drawbacks including limited scalability and the need for resin attachment and cleavage and off-column attachment. Importantly, the use of peptides risks an immune response to the conjugates, which would prevent repeated administrations for diseases requiring long-term treatment. To develop an efficient delivery composition which is costeffective, is not immunogenic, and is stable under physiological conditions, we sought to exploit nonlinear, nonpeptidic, and nonnatural architectures. Relatively few attempts have been made to use guanidine groups in branched or dendritic structures, but those attempts have produced some useful data that helped our development process. For example, polyguanidino dendrimers using triamine-based diamino acid monomeric units to form a series of octa-guanidine dendrimers show differences in uptake efficiency related to the structural flexibility of the dendritic architecture (28). Branched-chain arginine peptides translocate through cell membranes and bring exogenous proteins into cells; eight arginine residues was the number that exhibited the most

10.1021/bc8001437 CCC: $40.75  2008 American Chemical Society Published on Web 06/20/2008

In Vivo Delivery of Morpholino Antisense Oligo

efficient internalization (29). Dendritic oligoguanidines based on amino triol subunits translocate through the cell membrane (30). An alternative architecture based on an amino triacid scaffold demonstrated that dendritic molecular transporters can not only enable transport of bioactive cargo across the cell membrane, but can also direct the delivery into particular intracellular compartments (31). However, the reported syntheses of dendritic oligoguanidines are lengthy or involve expensive reagents for their assembly. We directed our efforts to developing a dendritic structure which mimics features of branched octaguanidines but provides cost-effective synthesis, convenient conjugation with bioactive molecules (Morpholino in this study), and superior performance in transporting compounds across biological barriers. For a molecular transporter to possess the aforementioned features, we proposed that the constitution of the dendritic scaffold should allow assembly of the oligoguanidine through a variant of a segment-multiplying strategy and had to allow attachment of a bioactive substance to its orthogonal functional site. We chose triazine as a trifunctional entity on which two sites could be used for branching side arms while a Morpholino oligo could be linked to its third functional site. To construct the molecular transporter in a cost-effective manner, we used diethanolamine to displace two chlorine atoms, yielding a tetraalcohol intermediate. After formation of a tetra-carbonate from the corresponding alcohols, a secondary amine from a triamine moiety in which the two terminal primary amines are in a protected form was contacted with the tetra-carbonate to give octa-amine, thus providing eight side chains each containing a terminal primary amino group in a protected form. To the third functional site of triazine, a reactive linking group which could conjugate with a Morpholino oligo was installed. Conjugation on synthesis resin allowed convenient removal of excess reagents, and the conjugate thus formed was cleaved from the synthesis resin by ammonia treatment. This ammonia treatment simultaneously generated an unprotected octa-amine intermediate which was subsequently converted to octa-guanidine by a single perguanidinylation step, yielding a conjugate of Morpholino with a transporter containing a triazine as the core scaffold (Scheme 1). These conjugates which are enabled for in ViVo delivery are referred to as “Vivo-Morpholinos”. We conjugated the transporter with a Morpholino oligo targeted to block a mutant RNA splicing site whose blockage leads to normal translation of an enhanced green fluorescent protein (EGFP) reporter. Using the signal from this reporter gene, relative in ViVo delivery efficacies were determined for a variety of tissues in transgenic mice.

Bioconjugate Chem., Vol. 19, No. 7, 2008 1465 Scheme 1. Structure of Vivo-Morpholino (VM), a Conjugate of Transporter Composition with a Morpholino Oligo

Scheme 2. Synthesis of Building Components for the Transporter Composition

EXPERIMENTAL PROCEDURES Materials and Methods. All reagents and solvents were purchased from commercial sources and were used as received. Components of the transporter were synthesized from readily available starting materials (Scheme 2). Analytical TLC was carried out on Baker-flex silica gel IB2-F and spots were located by UV illumination. Column chromatography was conducted through silica gel (60-200 mesh) from Fisher Scientific. All NMR spectra were measured on a 400 MHz Bruker FT-NMR spectrometer. MALDI-TOF MS were carried out on an Applied Biosystems Voyager using a matrix of 2′,4′,6′-trihydroxyacetophenone (THAP). Oasis HLB LP extraction cartridges (Waters Corporation, Milford, MA, USA) were used for purification of Vivo-Morpholino. Synthesis of N-Tritylpiperazine (1). Piperazine (107.5 g, 1.25 mol) was dissolved in dichloromethane (500 mL). Trityl chloride (69.7 g, 0.25 mol) was added to the mixture cooled in an ice bath. After addition, the mixture was kept at room temperature for 30 min. The mixture was washed with water

(500 mL, three times) and the organic layer was separated and dried over sodium sulfate. The solution was concentrated to ca. 100 mL and added to hexane (1 L). The solid was removed by filtration. The filtrate was evaporated to dryness to give a white solid (80 g, 97%). 1H NMR (400 MHz, CDCl3) δ ) 1.31 (br, 1H), 3.04 (br, 8H), 7.17-7.52 (m, 15H). Synthesis of Bis(trifluoroacetamidohexyl)amine (2). To a stirred solution of bis(hexamethylene)triamine (8.62 g, 40 mmol) in acetonitrile (120 mL) cooled in an ice bath was added water (0.864 mL) and ethyl trifluoroacetate (16.7 mL, 140 mmol). After addition, the mixture was refluxed for 16 h. The solvents were removed by evaporation. The residue solidified upon storing at room temperature and was used without further purification. Synthesis of Di(4-nitrophenyl) Suberate (3). Suberic acid (8.71 g, 50 mmol) and 4-nitrophenol (14.61 g, 105 mmol) were dissolved in dichloroethane (100 mL). 1,3-Diisopropylcarbo-

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diimide (16.28 mL, 104 mmol) was added to the mixture. The mixture was kept at room temperature for 16 h. The solid was removed by filtration. The filtrate was loaded on a silica gel column (silica gel 140 g), eluting with dichloromethane. After removal of the solvent, a white solid was obtained (18.4 g, 88%). 1 H NMR (400 MHz, CDCl3) δ ) 1.54 (m, 4H), 1.84 (m, 4H), 2.66 (t, 7.44 Hz, 4H), 7.31 (d, 9.39 Hz, 4H), 8.30 (d, 9.20 Hz, 4H). Synthesis of 2-(4-Tritylpiperazinyl)-4,6-bis(diethanolamino)triazine (4). Cyanuric chloride (922 mg, 5 mmol) was dissolved in a solution of tetrahydrofuran (10 mL) containing N,N-diisopropylethylamine (1.74 mL, 10 mmol). N-Tritylpiperazine (1) (1.642 g, 5 mmol) dissolved in tetrahydrofuran (10 mL) was added to the mixture cooled in an ice bath. The mixture was kept at 0 and 25 °C for 30 min, respectively. Diethanolamine (5 mL, 52 mmol) was added to the mixture. The mixture was heated at 70 °C for 16 h. The volatile materials were removed by evaporation. The residue was dissolved in ethyl acetate (200 mL) and washed with sodium bicarbonate (150 mL) and water (200 mL × 2) and dried over sodium sulfate. After removal of the solvent, a white solid was obtained (2.82 g, 92%). 1H NMR (400 MHz, CDCl3) δ ) 2.36 (br, 2H), 3.70 (br, 8H), 3.71 (br, 12H), 3.83 (br, 2H), 4.01 (br, 2H), 4.39 (br, 2H), 7.17-7.50 (m, 15H). Synthesis of 2-(4-Tritylpiperazinyl)-4,6-bis[di(4-nitrophenyloxycarbonyloxyethyl)amino]triazine (5). 2-(4-Tritylpiperazinyl)-4,6-bis(diethanolamino)triazine (4) (2.82 g, 4.59 mmol) was dissolved in acetone (40 mL). Triethylamine (1 mL) was added to the mixture, followed by bis(4-nitrophenyl) carbonate (8.4 g, 27.54 mmol). The mixture was kept at room temperature for 48 h. The solvent was then removed. The product was obtained by column purification to give a yellowish solid (4.8 g, 82%). 1H NMR (400 MHz, DMSO-d6) δ ) 2.45 (br, 2H), 3.94 (m, 14H), 4.51 (m, 8H), 6.87 (d, 9.19 Hz, 8H), 7.17-7.49 (m, 15H), 8.07 (d, 9.19 Hz, 8H). Synthesis of 2-(4-Tritylpiperazinyl)-4,6-bis{di[di(trifluoroacetamidohexyl)aminocarbonyloxyethyl]amino}triazine (6). 2-(4-Tritylpiperazinyl)-4,6-bis[di(4-nitrophenyloxycarbonyloxyethyl)amino]triazine (5) (2.03 g, 1.6 mmol) was dissolved in acetone (40 mL). N,N-Diisopropylethylamine (2.8 mL, 16 mmol) was added to the mixture, followed by bis(trifluoroacetamidohexyl)amine (2) (4.16 g, 8 mmol). The reaction mixture was kept at room temperature for 16 h. The volatile materials were removed by evaporation. The residue was chromatographed to give an oily foam (3.28 g, 87%). 1H NMR (400 MHz, DMSOd6) δ ) 1.21 (br, 32H), 1.42 (br, 32H), 3.07 (br, 8H), 3.15 (br, 32H), 3.72 (br, 8H), 4.08 (br, 8H), 7.19-7.45 (m, 15H), 9.35 (s, 8H). MALDI MS: calculated for C102H143F24N19O16, 2347.3; found, 2356.9. Synthesis of 2-[(4-Nitrophenyl)oxycarbonylhexamethylenecarbonylpiperazinyl]-4,6-bis{di[di(trifluoroacetamidohexyl)aminocarbonyloxyethyl]amino}triazine (8). 2-(4-Tritylpiperazinyl)-4,6-bis{di[di(trifluoroacetamidohexyl)aminocarbonyloxyethyl]amino}triazine (6) (1.0 g, 0.43 mmol) was dissolved in methanol (3.2 mL) and the solution was mixed with 5% cyanoacetic acid in 2,2,2-trifluoroethanol (5 mL). The mixture was kept at room temperature for 10 min. The solvents were removed by evaporation. The residue was then diluted with dichloromethane (50 mL) and washed with saturated sodium bicarbonate (30 mL). The organic layer was separated and dried over sodium sulfate. After removal of the solvent, the crude product 7 was dissolved in acetone (10 mL). N,N-Diisopropylethylamine (0.4 mL, 2.3 mmol) was added to the mixture, followed by di(4-nitrophenyl) suberate (3) (732 mg, 1.76 mmol). The reaction mixture was kept at 50 °C for 2 h. The solvents were removed and the product was isolated from silica gel column chromatography to give an oily paste (0.95 g, 93%).

Li and Morcos

H NMR (400 MHz, DMSO-d6) δ ) 1.23 (br, 34H), 1.45 (br, 34H), 1.54 (m, 2H), 1.67 (m, 2H), 2.35 (t, 7.23 Hz, 2H), 2.64 (t, 7.43 Hz, 2H), 3.15 (m, br, 32H), 3.47 (br, 4H), 3.64 (br, 2H), 3.69 (br, 2H), 3.76 (br, 8H), 4.14 (br, 8H), 7.44 (d, 9.19 Hz, 2H), 8.31 (d, 9.19 Hz, 2H), 9.36 (s, br, 8H). MALDI MS: calculated for C97H144F24N20O21, 2382.3; found, 2390.6. General Procedure for Synthesis of Vivo-Morpholino 11, a Conjugate Containing the Transporter Composition and a Morpholino Antisense Oligo. The precursor transporter composition 8 (50 mg) in 1,3-dimethyl-2-imidazolidinone (0.18 mL) containing 5% of 1-hydroxybenzotriazole as catalyst and 5% of triethylamine was incubated with Morpholino (300 nmol) at 60 °C for 2 h. After removal of the solvent, concentrated ammonia (0.30 mL) was added and the mixture was incubated at 50 °C for 5 h. 18% ammonia (0.30 mL) was added to the mixture, followed by O-methylisourea hydrochloride (100 mg). The mixture was incubated at 65 °C for 45 min. Water was added to dilute the mixture, and the product was isolated by using Oasis HLB LP extraction cartridge to give VivoMorpholino. Functional Quantitative Assessment of Delivery in ViWo of a Conjugate Comprising the Transporter Composition with a Morpholino. Intravenous injections of Vivo-Morpholino were carried out in Ryszard Kole’s laboratory by Peter Sazani using transgenic mice that ubiquitously express a modified enhanced green fluorescent protein (EGFP) pre-mRNA containing an aberrantly spliced β-globin intron (IVS2-654) (32). The Vivo-Morpholino oligo called VM654-25 with the sequence 5′-TTGCTATTACCTTAACCCAGAAATT was injected i.v. into mice at 12.5 mg/kg for 4 consecutive days, sacrificed on day 5, and analyzed as described previously (32).

1

RESULTS AND DISCUSSION We set out to design a transporter composition which could be constructed very efficiently. Therefore, many factors such as scaffold core selection, functional group multiplication, orthogonal protecting group manipulation, and methods for formation of guanidine headgroups had to be taken into consideration for overall economy of process. We chose triazine architecture since it provides three functional sites, one for installing a linking group to covalently conjugate with a Morpholino oligo and the other two for anchoring the dendritic arms. Importantly for the selection of triazine, it is unnatural and robust in the living system. This increases the likelihood of avoiding problems such as antigenicity and toxicity and improving properties such as bioavailability, stability, and safety for use in ViVo. The synthesis uses readily available starting materials (Scheme 2). Monotritylated piperazine 1 is obtained by treating trityl chloride with an excess amount of piperazine. Reaction of bis(hexamethylene)triamine with ethyl trifluoroacetate selectively protects the two terminal primary amines and gives the intact secondary amine 2. Homobifunctional linker 3 is obtained by formation of the nitrophenyl ester of suberic acid. The whole scheme for assembly of Vivo-Morpholino is illustrated in Scheme 3. The protected piperazine 1 reacts with cyanuric chloride to give monosubstituted dichlorotriazine. The dichloride is then treated with diethanolamine to give tetraalcohol 4. The hydroxyl groups are activated with bis(4nitrophenyl) carbonate to provide tetra-carbonate 5. The tetra-carbonate 5 is treated with a secondary amine 2 to form tetra-carbamate 6 containing octa-amine in a protected form. The secondary amine of piperazine 7 is regenerated by acidic deprotection, which reacts with the homobifunctional linker 3 to give the transporter moiety 8 with an active functional moiety at one terminus. This activated ester functional group 8 couples with a Morpholino oligo on the synthesis resin to give conjugate

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Scheme 3. Synthetic Scheme for Assembly of Vivo-Morpholino

9. Exposure of 9 to concentrated ammonia cleaves the conjugate from the synthesis resin and removes all the protecting groups on the Morpholino oligo bases and the protecting groups on the octa-amines to give conjugate 10. Perguanidinylation with O-methylisourea quantitatively converts all the amino groups on the dendritic scaffold to guanidine headgroups, furnishing the assembly of Vivo-Morpholino 11.

Triazine Core and Functional Group Multiplication. Dendrimers represent an attractive transporter scaffold offering economical assembly of oligoguanidine transporters through a segment-multiplying strategy. The ease of displacement of chloride atoms in cyanuric chloride by various nucleophiles in the presence of a hydrochloride acceptor (usually tertiary amines) makes this reagent useful for preparation of mono-,

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Li and Morcos

Figure 1. Densitometric analysis of RT-PCR transcripts of splice correction by Vivo-Morpholino in comparison with bare Morpholino. The data is collected from four mice, two injected with Vivo-Morpholino and two injected with bare Morpholino at 12.5 mg/kg/day i.v. for four days, followed by sacrifice and RT-PCR one day after the final injection.

Figure 2. Assessment of liver toxicity. No significant changes in aspartate aminotransferase (AST) or alanine aminotransferase (ALT) levels in the liver were detected with Vivo-Morpholinos delivered at 12.5 mg/kg/day i.v. for four days. Data shown as fold AST or ALT over prebleed, one day after final injection.

di-, and trisubstituted 1,3,5-triazine (33). The substitution of chloride can be controlled by temperature to run in a stepwise manner. In this study, the first amino group from N-tritylpiperazine 1 was introduced to give monosubstituted triazine at 0 °C in the presence of N,N-diisopropylethylamine as base. The other two chloride atoms of the triazine were displaced at 70 °C by diethanolamine which was used in excess as base to neutralize the hydrogen chloride generated. The trityl group serves as the protecting group which, after removal at a later stage, generates an amino group for installing an active linking group to be used for conjugation with a Morpholino oligo. The formation of this intermediate provides the core scaffold on which the tetra-alcohol 4 not only extended the side-arms and doubled the number of the functional groups, but also paved the way for further multiplication of side arms. We activated the hydroxyl groups to form reactive carbonate 5. By contacting a secondary amine of a triamine moiety in which the two terminal primary amines are in a protected form, the tetracarbonate 5 yielded an eight side chain entity 6, each of which contains a terminal protected primary amine which forms a guanidine headgroup after deprotection and guanidinylation. Protecting Group Manipulation. We chose bis(hexamethylene)triamine so that its secondary amine, after selective protection of two terminal primary amines, could contact reactive carbonate 5 to form carbamate 6 and double the number of side arms. The methylene units of this commercially available triamine link and spatially separate the three nitrogen atoms. After the primary amino groups are converted to guanidine headgroups, the side arms are flexibly spaced which could be beneficial for efficient contact with cell membrane surface and

consequently efficient intracellular uptake. Trifluoroacetyl is selected for protecting terminal primary amines, based on the following four critical reasons: (a) Since methyl or ethyl trifluoroacetate are mild and selective reagents to protect primary amines in the presence of secondary amines (34), the secondary amine can remain intact while the primary amines are protected so that the secondary amine can be used to contact the carbonate 5. (b) An orthogonal strategy has to be used since a different amino group must be selectively deprotected for ultimate conjugation with a Morpholino oligo. Acid-labile trityl group is selected for this amino group since trifluoroacetyl group is labile toward basic conditions but stable toward acidic conditions. (c) Trifluoroacetyl protecting groups on the amines can be cleaved by ammonolysis, a condition previously serving the double duty of removing protecting groups on Morpholino oligo bases and cleaving the oligo from the synthesis resin. Therefore, exposure of ammonia fits into the normal Morpholino synthesis process. This strategy proved very useful since the coupling of the transporter composition can be carried out with Morpholino antisense oligo while it is still on the synthesis resin, and subsequent ammonolytic treatment not only removes all the protecting groups on the Morpholino oligo and the oligoamine moiety, but also cleaves the Morpholino with its conjugated precursor transporter composition from the synthesis resin. The advantage of this triple duty maneuver is simple and economic production. (d) Ammonia is also a basic reagent for converting an amino group to a guanidine in the presence of a guanidinylation agent, O-methylisourea hydrochloride. Therefore, without any purification, the ammonia used for cleavage from resin and deprotection of protecting groups of the Morpholino oligo bases and the dendrimeric amine can be carried over for the subsequent guanidinylation. By choosing the selected protecting group (trifluoroacetyl) and the selected protecting group removal agent (ammonia), the whole process for producing a conjugate containing a Morpholino oligo and transporter composition is significantly simplified. Perguanidinylation. In principle, guanidino groups can be installed before the whole transporter composition is conjugated with a Morpholino oligo. However, because of the nature of the guanidine moiety, it requires expensive reagents for the conversion from primary amines and for the protection of the guanidines thus formed. In this study, we adopted a postperguanidinylation performed in solution phase after deprotection and cleavage of the conjugate from the solid support with

In Vivo Delivery of Morpholino Antisense Oligo

ammonia. Without any purification, the mixture thus obtained was treated further with O-methylisourea hydrochloride, giving Vivo-Morpholino, the conjugate of transporter composition with a Morpholino oligo. O-Methylisourea hemisulfate could also be used for the conversion, but side products corresponding to the incomplete guanidinylation were observed, which was in agreement with the reported results (35). We did not find any side reactions toward the other amino groups present in the bases of Morpholino oligo under the guanidinylation conditions. This off-resin method to convert amino functions quantitatively into guanidines proves a concise and straightforward means to obtain the target compounds. Systemic Delivery of Vivo-Morpholino in Transgenic Mice. Kole and co-workers have developed a strain of transgenic mice modeled after their test system used in cell culture (36) but replacing the luciferase component with enhanced green fluorescent protein (EGFP) (37). That RNA transcript, expressed in all tissues of the mice, contains a globin intron mutation that causes a splicing error which prevents expression of the EGFP. Annealing an appropriate Morpholino antisense oligo to the mutant splice site corrects the splicing error and results in the expression of EGFP. Thus, the technology of visualizing green fluorescence in a specific tissue or evaluating the corrected transcript by RT-PCR has been used to rigorously assess cytosolic delivery into cells of that tissue in ViVo. The ability of Vivo-Morpholinos to achieve cytosolic delivery in ViVo was assessed by administering a Vivo-Morpholino targeting the splice mutation site. This was introduced by intravenous injection at 12.5 mg/kg for 4 consecutive days into two of the transgenic mice harboring the splice mutation, followed by sacrifice on day 5. Delivery was assessed by densitometric RT-PCR analysis of RNA transcripts isolated from various tissues (Figure 1). Nearly complete splice correction was observed in liver, small intestine, colon, skeletal muscle, diaphragm, and kidney, indicating excellent delivery with VivoMorpholinos. Delivery into the spleen and stomach was also high, although not sufficient for complete splice correction at the concentration tested. Modest delivery was achieved in lung, heart, skin, and brain. The AST-ALT data were also collected (Figure 2), indicating that the Vivo-Morpholinos were not toxic to the liver in mice at 12.5 mg/kg over 4 days. These striking results show that Vivo-Morpholinos are capable of achieving a quantifiable level of delivery in all tissues tested from repeated i.v. injections. This suggests that Vivo-Morpholinos are both achieving a high level of access to the cytosol of cells within tissues and able to avoid the inhibitory effects of serum and other cellular components.

CONCLUSION In this report, we described synthesis of a novel dendritic molecular transporter and its conjugate with a Morpholino oligo and investigated intracellular uptake of the conjugate in transgenic mice by quantitative functional assessment. The transporter composition is a compact, biocompatible dendritic scaffold with guanidine headgroups spaced in a flexible framework. The assembly of the core structure exploits trifunctional triazine both for installing a linking site for ultimate conjugation with a Morpholino oligo and for anchoring the side arms which were then multiplied to extend the number of side chains. This structure was demonstrated to provide efficient delivery into cells of transgenic mice with minimal toxicity. Further investigations will involve the studies of tissue distribution, biological stability, and pharmacokinetics of delivery-enabled Morpholino oligos. Our present results show that the synthetic approach of this molecular transporter and its highly efficient delivery ability have advanced Morpholino antisense technology into a stage

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where cost-effective production of delivery-enhanced Morpholino oligos for studies in living animals is now practical.

ACKNOWLEDGMENT We would like to thank Dr. James E. Summerton for his insightful discussion. Mrs. Chandell L. Terwilliger and Mrs. Jeannine M. Turner are thanked for their analytical assistance. We are grateful to Dr. Jon D. Moulton for his critical proofreading of the manuscript.

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