Nicholas J. Baumhover

Synthesis and radiolabeling of chelator-RNA aptamer bioconjugates with copper-64 for targeted molecular imaging.

Ribonucleic acid (RNA) aptamers with high affinity and specificity for cancer-specific cell-surface antigens are promising reagents for targeted molecular imaging of cancer using positron emission tomography (PET). For this application, aptamers must be conjugated to chelators capable of coordinating PET-radionuclides (e.g., copper-64, (64)Cu) to enable radiolabeling for in vivo imaging of tumors. This study investigates the choice of chelator and radiolabeling parameters such as pH and temperature for the development of (64)Cu-labeled RNA-based targeted agents for PET imaging. The characterization and optimization of labeling conditions are described for four chelator-aptamer complexes. Three commercially available bifunctional macrocyclic chelators (1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid mono N-hydroxysuccinimide [DOTA-NHS]; S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid [p-SCN-Bn-NOTA]; and p-SCN-Bn-3,6,9,15-tetraazabicyclo [9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid [p-SCN-Bn-PCTA]), as well as the polyamino-macrocyclic diAmSar (3,6,10,13,16,19-hexaazabicyclo[6.6.6] icosane-1,8-diamine) were conjugated to A10-3.2, a RNA aptamer which has been shown to bind specifically to a prostate cancer-specific cell-surface antigen (PSMA). Although a commercial bifunctional version of diAmSar was not available, RNA conjugation with this chelator was achieved in a two-step reaction by the addition of a disuccinimidyl suberate linker. Radiolabeling parameters (e.g., pH, temperature, and time) for each chelator-RNA conjugate were assessed in order to optimize specific activity and RNA stability. Furthermore, the radiolabeled chelator-coupled RNA aptamers were evaluated for binding specificity to their target antigen. In summary, key parameters were established for optimal radiolabeling of RNA aptamers for eventual PET imaging with (64)Cu.

Synthesis and in vitro testing of new potent polyacridine-melittin gene delivery peptides.

The combination of a polyacridine peptide modified with a melittin fusogenic peptide results in a potent gene transfer agent. Polyacridine peptides of the general formula (Acr-X)(n)-Cys were prepared by solid-phase peptide synthesis, where Acr is Lys modified on its epsilon-amine with acridine, X is Arg, Leu, or Lys and n is 2, 3, or 4 repeats. The Cys residue was modified by either a maleimide-melittin or a thiolpyridine-Cys-melittin fusogenic peptide resulting in reducible or non-reducible polyacridine-melittin peptides. Hemolysis assays established that polyacridine-melittin peptides retained their membrane lytic potency relative to melittin at pH 7.4 and 5. When combined with plasmid DNA, the membrane lytic potency of polyacridine-melittin peptides was neutralized. Gene transfer experiments in multiple cell lines established that polyacridine-melittin peptides mediate expression as efficiently as PEI. The expression was very dependent upon a disulfide bond linking polyacridine to melittin. The gene transfer was most efficient when X is Arg and n is 3 or 4 repeats. These studies establish polyacridine peptides as a novel DNA binding anchor peptide.

Metabolically stabilized long-circulating PEGylated polyacridine peptide polyplexes mediate hydrodynamically stimulated gene expression in liver.

A novel class of PEGylated polyacridine peptides was developed that mediate potent stimulated gene transfer in the liver of mice. Polyacridine peptides, (Acr-X)n-Cys-polyethylene glycol (PEG), possessing 2–6 repeats of Lys-acridine (Acr) spaced by either Lys, Arg, Leu or Glu, were Cys derivatized with PEG (PEG5000 kDa) and evaluated as in vivo gene transfer agents. An optimal peptide of (Acr-Lys)6-Cys-PEG was able to bind to plasmid DNA (pGL3) with high affinity by polyintercalation, stabilize DNA from metabolism by DNAse and extend the pharmacokinetic half-life of DNA in the circulation for up to 2 h. A tail vein dose of PEGylated polyacridine peptide pGL3 polyplexes (1 μg in 50 μl), followed by a stimulatory hydrodynamic dose of normal saline at times ranging from 5 to 60 min post-DNA administration, led to a high level of luciferase expression in the liver, equivalent to levels mediated by direct hydrodynamic dosing of 1 μg of pGL3. The results establish the unique properties of PEGylated polyacridine peptides as a new and promising class of gene delivery peptides that facilitate reversible binding to plasmid DNA, protecting it from DNase in vivo resulting in an extended circulatory half-life, and release of transfection-competent DNA into the liver to mediate a high-level of gene expression upon hydrodynamic boost.

PEG length and chemical linkage controls polyacridine peptide DNA polyplex pharmacokinetics, biodistribution, metabolic stability and in vivo gene expression.

The pharmacokinetics (PK), biodistribution and metabolism of non-viral gene delivery systems administered systemically are directly related to in vivo efficacy. The magnitude of luciferase expression in the liver of mice following a tail vein dose of a polyplex, composed of 1 μg of pGL3 in complex with a polyethylene glycol (PEG) polyacridine peptide, followed by a delayed hydrodynamic (HD) stimulation (1-9 h), depends on the HD stimulation delay time and the structure of the polyacridine peptide. As demonstrated in the present study, the PEG length and the type of chemical linkage joining PEG to the polyacridine peptide dramatically influence the in vivo gene transfer efficiency. To understand how PEG length, linkage and location influence gene transfer efficiency, detailed PK, biodistribution and HD-stimulated gene expression experiments were performed on polyplexes prepared with an optimized polyacridine peptide modified through a single terminal Cys or Pen (penicillamine) with a PEG chain of average length of 2, 5, 10, 20, or 30 kDa. The chemical linkage was examined by attaching PEG(5 kDa) to the polyacridine peptide through a thiol-thiol (SS), thiol-maleimide (SM), thiol-vinylsulfone (SV), thiol-acetamide (SA), penicillamine-thiol-maleimide (PM) or penicillamine-thiol-thiol (PS). The influence of PEG location was analyzed by attaching PEG(5 kDa) to the polyacridine peptide through a C-terminal, N-terminal, or a middle Cys residue. The results established rapid metabolism of polyplexes containing SV and SA chemical linkages that leads to a decreased polyplex PK half-life and a complete loss of HD-stimulated gene expression at delay times of 5 h. Conversely, polyplexes containing PM, PS, and SM chemical linkages were metabolically stable, allowing robust HD-stimulated expression at delay times up to 5h post-polyplex administration. The location of PEG(₅ kDa) within the polyacridine peptide exerted only a minor influence on the gene transfer of polyplexes. However, varying the PEG length from 2, 5, 10, 20, or 30 kDa dramatically altered polyplex biodistribution, with a 30 kDa PEG maximally blocking liver uptake to 13% of dose, while maintaining the ability to mediate HD-stimulated gene expression. The combination of results establishes important relationships between PEGylated polyacridine peptide structure, physical properties, in vivo metabolism, PK and biodistribution resulting in an optimal PEG length and linkage that leads to a robust HD-stimulated gene expression in mice.

Discovery of Metabolically Stabilized Electronegative Polyacridine-PEG Peptide DNA Open Polyplexes

Cationic condensing peptides and polymers bind electrostatically to DNA to form cationic polyplexes. While many cationic polyplexes are able to achieve in vitro transfection mediated through electrostatic interactions, few have been able to mediate gene transfer in vivo. The present study describes the development and testing of polyacridine PEG-peptides that bind to plasmid DNA by intercalation resulting in electronegative open polyplex DNA. Polyacridine PEG-peptides were prepared by chemically conjugating 6-(9-acridinylamino) hexanoic acid onto side chains of Lys in PEG-Cys-Trp-(Lys)(3, 4, or 5). The resulting PEG-Cys-Trp-(Lys-(Acr))(3, 4, or 5) peptides bound tightly to DNA by polyintercalation, rather than electrostatic binding. Unlike polycationic polyplexes, polyacridine PEG-peptide polyplexes were anionic and open coiled, as revealed by zeta potential and atomic force microscopy. PEG-Cys-Trp-(Lys-(Acr))(5) showed the highest DNA binding affinity and the greatest ability to protect DNA from metabolism by DNase. Polyacridine PEG-peptide DNA open polyplexes were dosed intramuscularly and electroporated in mice to demonstrate their functional activity in gene transfer. These results establish polyacridine PEG-peptide DNA open polyplexes as a novel gene delivery method for in vivo use.

The uptake mechanism of PEGylated DNA polyplexes by the liver influences gene expression

Two uptake mechanisms were identified for PEGylated DNA polyplex biodistribution to the liver. At a low polyplex dose, a rapid-uptake mechanism dominates, resulting in 60% capture by liver in 5 min, due to a saturable receptor-mediated process. Rapid-uptake led to the fast metabolism of polyplexes by liver (t1/2=2.1 h), correlating with a 1-μg pGL3 polyplex dose losing full transfection competency after 4 h in the liver. Dose escalation of either polyplex or poly(ethylene glycol) (PEG) peptide led to the saturation of rapid-uptake and revealed a delayed-uptake mechanism for polyplexes by liver. Delayed-uptake was characterized by the slower liver accumulation of 40% of the polyplex dose over 40 min, followed by slow metabolism (t1/2=15 h) and an extended time (12 h) for a 1-μg pGL3 polyplex dose, remaining fully transfection competent in the liver. The delayed-uptake mechanism is consistent with polyplexes crossing liver fenestrated endothelial cells to reach steady state in the space of Disse. The results describe how to control polyplex biodistribution to liver to avoid rapid-uptake and metabolism, in favor of delayed-uptake, to preserve polyplex transfection competency in the liver for up to 12 h.

Structure-Activity Relationship of PEGylated Polylysine Peptides as Scavenger Receptor Inhibitors for Non-Viral Gene Delivery.

PEGylated polylysine peptides of the general structure PEG30 kDa-Cys-Trp-LysN (N = 10 to 30) were used to form fully condensed plasmid DNA (pGL3) polyplexes at a ratio of 1 nmol of peptide per μg of DNA (ranging from N:P 3:1 to 10:1 depending on Lys repeat). Co-administration of 5 to 80 nmols of excess PEG-peptide with fully formed polyplexes inhibited the liver uptake of (125)I-pGL3-polyplexes. The percent inhibition was dependent on the PEG-peptide dose and was saturable, consistent with inhibition of scavenger receptors. The scavenger receptor inhibition potency of PEG-peptides was dependent on the length of the Lys repeat, which increased 10-fold when comparing PEG30 kDa-Cys-Trp-Lys10 (IC50 of 20.2 μM) with PEG30 kDa-Cys-Trp-Lys25 (IC50 of 2.1 μM). We hypothesize that PEG-peptides inhibit scavenger receptors by spontaneously forming small 40 to 60 nm albumin nanoparticles that bind to and saturate the receptor. Scavenger receptor inhibition delayed the metabolism of pGL3-polyplexes, resulting in efficient gene expression in liver hepatocytes following delayed hydrodynamic dosing. PEG-peptides represent a new class of scavenger inhibitors that will likely have broad utility in blocking unwanted liver uptake and metabolism of a variety of nanoparticles.

163. Hepatocyte Targeted Endosomal Escape Agents

The successful development of potent non-viral DNA and mRNA delivery agents to transfect liver hepatocytes in vivo requires the use of an endosomal escape molecule. Two major strategies have emerged, one that utilizes membrane lytic peptides, and a second that uses proton sponge polymers. Both approaches would require administration of excess endosomal release agent with a smaller dose of targeted polyplex. The present study compared these approaches by conducting 384-well in vitro transfections on primary hepatocytes, as previously described1. An N-glycan targeted melittin was designed to target hepatocytes via the asialoglycoprotein receptor. The synthesis used a purified triantennary N-glycan from bovine fetuin as an potent high affinity asialoglycoprotein receptor targeting ligand that was conjugated by disulfide bound to an N-terminal Cys on melittin, a 26 amino acid membrane lytic peptide. Following targeted entry into hepatocytes via receptor mediated endocytosis, melittin is bioactivated by reduction of the disulfide bond to release the N-glycan, leading to endosomal lysis. RBC hemolysis assays established the membrane lytic activity of triantennary-melittin was muted prior to reduction, which allowed it to be safely i.v. dosed in mice. A second strategy involved the modification of PEI by reductive amination with lactose followed by acetylation, resulting in an asialoglycoprotein receptor targeted proton sponge polymer. The resulting LacAcyl-PEI was purified by RP-HPLC and characterized by NMR. The i.v. dosing of LacAcyl PEI in mice demonstrated that it possessed a much greater safety margin than unmodified PEI. These novel endosomal escape agents are being compared for their ability to mediate in vitro gene transfer of glycan targeted DNA and mRNA in miniaturized primary hepatocyte transfection assays.

271. Improved Gene Transfer with a New Class of PEG-Peptide Scavenger Receptor Inhibitors

Scavenger receptors (SR) found on Kupffer and fenestrated endothelial cells of the recticuloendothelial system (RES) in liver are responsible for the capture and degradation of both viral1 and non viral gene delivery systems 2. SR inhibition using polyinosinic acid (Poly-I) has been shown to improve the gene transfer efficiency of AdV, AAV, and measles virus by blocking rapid metabolism in the liver. While Poly-I is reportedly not toxic to mice, co-administration of Poly-I with AdV decreases the lethal threshold for AdV in mice by an unknown mechanism1, making Poly-I inhibition of SR a clinically unacceptable approach to inhibit viral uptake by the RES in the liver. In the present study, we report the discovery of a new class of PEGylated polylysine peptides that are potent and safe SR inhibitors. PEGylated(30kDa)-Cys-Trp-Lys(N) (where N = 10, 15, 20, 25, and 30) were prepared by solid phase peptide synthesis and administered to mice i.v. with 125I-pGL3. Administration of 1 nmol of PEG-peptide with 1 mg of 125I-pGL3 resulted in capture of 60% of the polyplex dose by the liver. However, dose-escalation of the PEG-peptide revealed a dose-dependent decrease in 125I-pGL3 PEG-peptide polyplex capture by the liver, resulting from SR inhibition. A maximum dose of 80 nmols of PEG(30kDa)-Cys-Trp-Lys25 inhibited SR mediated uptake of 125I-pGL3 by the liver to 10%. The potency of PEG-peptide inhibition of SR was also dependent on the length of the Lys repeat, with Lys30kDa=Lys25Lys20Lys15 Lys10. Application of delayed hydrodynamic (HD)-stimulation at 1 hour post-pGL3 polyplex delivery resulted in potent gene expression only when administering a SR inhibitory dose (80 nmol) of PEG-peptide. The reported PEG-peptides are proposed to function by in situ formation of 30 nm albumin nanoparticles in the blood that bind to SRs. This new class of safe and potent SR inhibitors may have broad applications by improving both viral and non-viral delivery systems by blocking SR-mediated uptake in the liver.