gene therapy protocols, 2nd

M E T H O D S I N M O L E C U L A R M E D I C I N E TM Gene Therapy Protocols Second Edition Edited by Jeffrey R Morgan Humana Press Poly-L-Lysine-Based Gene Delivery Systems 1 Poly-L-Lysine-Based Gene Delivery Systems Synthesis, Purification, and Application Charles P Lollo, Mariusz G Banaszczyk, Patricia M Mullen, Christopher C Coffin, Dongpei Wu, Alison T Carlo, Donna L Bassett, Erin K Gouveia, and Dennis J Carlo Introduction Nonviral gene delivery has great potential for replacement of recombinant protein therapy In many cases, gene therapies would be a considerable improvement over existing therapies because of putative advantages in dosing schedule, patient compliance, toxicity, immunogenicity, and cost Development of a nonviral gene delivery vehicle capable of efficient, cell-specific delivery will be a valuable addition to the clinical armamentarium The current situation has led to a focus on increasingly complex delivery systems as investigators try to achieve the delivery efficiency that viral systems already demonstrate It will be very difficult to create a self-assembling gene delivery system that incorporates molecular mechanisms similar to those that allow viruses to trespass on vascular, cellular, and intracellular barriers and effectively deliver viral DNA to the nucleus of mammalian cells However, much progress has been made with regard to production of uniform particles Steric stabilization of materials in vascular compartments has been an area of intense investigation, and numerous strategies for surface modification of delivery vehicles have shown positive effects (1–6) Incorporation of molecular components to accomplish receptor-mediated targeting, endosomal escape, and nuclear transport have all been attempted, with some success in vitro (7,8) From: Methods in Molecular Medicine, Vol 69, Gene Therapy Protocols, 2nd Ed Edited by: J R Morgan © Humana Press Inc., Totowa, NJ Lollo et al Fig Sample grafts 1.1 Poly-L-Lysine Poly-L-lysine (PLL) is a linear, biodegradable polymer that can be readily modified with a variety of chemical reagents to create novel conjugates with enhanced characteristics over those present in PLL per se In the gene delivery arena, researchers have typically tried to mimic characteristics of proteins that enable viruses to deliver their DNA or RNA payload so efficiently Thus, many synthetic chemists have focused on incorporating moieties that can facilitate cell-specific targeting, membrane penetration, and nuclear transport Another common synthetic goal is to modify PLL so that it can protect the DNA payload effectively More specifically, the intent is to diminish deleterious in vivo interactions such as immunogenicity, toxicity, adventitious binding, and uptake by the reticuloendothelial system PLL can be grafted with various agents to alter polyplex performance characteristics depending on desired outcome and area of investigation Cationic polymers other than PLL have also been modified and characterized in a similar fashion (9–11) 1.2 Grafting Grafts can consist of any natural or synthetic polymer, linear or branched, cyclic, heterocyclic, containing heteroatoms, or any combination of grafting molecules The number of grafted chains can be varied to suit specific applications (Fig 1) Poly-L-Lysine-Based Gene Delivery Systems Fig Grafting of receptor ligands onto a cationic polymer Fig Reaction of an activated ester with an amino group An amide bond-linked conjugate is produced 1.3 Ligand To achieve cell-specific targeting, receptor ligands can be grafted onto PLL or other cationic polymers (12–14) The preferred position of a ligand is on the exterior surface to ensure proper ligand recognition However, it is conceivable that ligands may also be partially buried and subject to molecular mechanisms that expose them at an appropriate time (15) Polymers, like polyethylene glycol (PEG), that are grafted onto surfaces form statistical clouds that are continually in flux Therefore, simple covalent attachment of ligand onto the terminal end of a polymeric chain does not guarantee ligand recognition The linker polymer, graft density, and chemistry will probably have to be optimized for individual cases (Fig 2) 1.4 Graft Attachment Nucleophilic substitution of activated esters is the most common chemistry to graft polymeric chains onto amino groups of proteins, cationic polymers, or more specifically PLL (16) The reaction of an activated ester with an amino group produces an amide bond-linked conjugate and results in a net loss of charge on the conjugate (Fig 3) This loss of positive charge along the polymer chain significantly weakens the binding of conjugate to DNA Conversely, chemistry that preserves the charge of the cationic domain is expected to have a lessened impact on DNA Lollo et al Fig Reaction of an electrophilic reagent with an ε-amino group of PLL binding since the binding will be affected only by steric hindrance generated from the grafted moieties For synthesis of our conjugates, we have chosen chemistries that preserve charges on the cationic domain and typically produce secondary and tertiary amines, and rarely quaternary ammonium species All these amine species bear a positive charge at physiologic pH and consequently will bind to DNA electrostatically The first method described below uses PEG–epoxide as the electrophilic reagent that reacts with ε-amino groups of PLL The product of the reaction is a secondary amine with a racemic βhydroxyl group (Fig 4) Grafts can be added successively if more than one feature is desired Alternatively, the grafting molecule can be engineered to contain more than one functional domain 1.5 Conjugate Synthesis, Purification, and Characterization A variety of grafted PLL conjugates have been successfully synthesized (17) These copolymers (e.g., poly-L-lysine-graft-R1-graft-R2-graft-R3) can have a variety of molecules grafted on amino groups of cationic polymers in a stepwise synthesis For example, PEG molecules can be grafted first (R1), followed by introduction of other functional groups such as ligands (R2), and finally fluorescent tags or other delivery-enhancing moieties (R3) The synthesis of one grafted copolymer is described below in stepwise fashion The procedure can be repeated to add other grafted domains Materials 2.1 Chemicals 10 Phosphate (J.T Baker, Phillipsburg, NJ) SP Sepharose FF resin (Amersham Pharmacia, Uppsala, Sweden) NaOH (J.T Baker) NaCl (J.T Baker) PLL 10K (Sigma, St Louis, MO) Lithium hydroxide monohydrate (E.M Science, Gibbstown, NJ) Methanol (VWR Scientific Products, West Chester, PA) BioCad 700E HPLC (PE Biosystems, Foster City, CA) UV/VIS detector (PE Biosystems) Glacial acetic acid (J.T Baker) Poly-L-Lysine-Based Gene Delivery Systems 11 12 13 14 15 16 17 18 19 20 PEG5K-epoxide (Shearwater Polymers, Huntsville, AL) Sephadex G-25 fine resin (Amersham Pharmacia) Trilactosyl aldehyde (Contract synthesis, e.g., SRI International, Menlo Park, CA) Amino-PEG3.4k-amino-tBOC (Shearwater Polymers) Sodium cyanoborohydride (Alfa Aesar, Ward Hill, MA) Methyl iodide (Aldrich, Milwaukee, WI) Trifluoroacetic acid (J.T Baker) Methylene chloride (VWR Scientific Products) Succinimidyl bromoacetate (Molecular Biosciences, Boulder, CO) Acetonitrile (J.T Baker) 2.2 Materials for DNA Manipulation Tris(hydroxymethyl)aminomethane (J.T Baker) EDTA (J.T Baker) Ethidium bromide (Sigma) 2.3 Materials for Animal Studies Ketamine (Phoenix Pharmaceuticals, St Joseph, MO) Xylazine (Phoenix Pharmaceuticals) Acepromazine (Fermenta Vet Products, Kansas City, MO) Potassium phosphate (J.T Baker) Triton X-100 (VWR Scientific Products) Sigma Firefly luciferase L-5256 (BD Pharmingen, San Diego, CA) 15-mL dounce homogenizer (Wheaton, Millville, NJ) Methods 3.1 Synthesis of Poly-L-Lysine-graft-R1-graft-R2-graft-R3 Copolymers R1 means PEG derivative and R2 and R3 no PEG derivative PLL-graft-PEG polymers can be prepared by reaction of a PEG-electrophile with ε-NH2 lysine groups under basic conditions For any specific copolymers, the ratio of activated PEG to poly-L-lysine, PEG size, and poly-L-lysine size can be varied as needed Poly-L-lysine 10K (600 mg, 0.06 mmol) and lithium hydroxide monohydrate (41 mg, 2.9 mmol) are dissolved in water (2 mL) and methanol (6 mL) in a siliconized glass flask Solid PEG5K-epoxide (600 mg, 0.12 mmol) is added to the flask, which is then sealed, and the solution is incubated at 65°C for 48 h After incubation, the solvent is removed in vacuo The product is redissolved in a loading buffer (0.1 M sodium phosphate, pH 6, in 10% MeOH [v/v]) The solution is loaded on a cation exchange column (SP Sepharose FF resin) attached to a high-performance liquid chromatography (HPLC) device (e.g., BioCad 700E), followed by an extensive washing step (up to 10 column volumes) Lollo et al The product is eluted with 0.1 N NaOH in 10% MeOH solution An in-line 214nm UV/VIS detector is used to monitor the eluant, and fractions are collected in a standard manner Fractions containing the product are combined and neutralized, and the solvent is removed in vacuo The dried product, which contains inorganic salts, is redissolved in a minimum amount of 0.05 M acetic acid in 30% MeOH solution and separated over a G-25 column (Amersham Pharmacia Sephadex G-25 fine resin) using the same acetic acid solution The fractions are pooled and lyophilized The average number of PEG moieties grafted onto each poly-L-lysine chain can be determined by 1H nuclear magnetic resonance (NMR) (18) 3.2 Synthesis of PL26k-graft-(ε-NH-CH2CO-NH-PEG-εTrilactose-Ligand)2.5 Stepwise grafting is one of the simplest ways to modulate properties of resulting copolymers However, it does not provide easy means of incorporating targeting moieties at their optimal positions The linker bearing the ligand should be at least as long (or longer) as other components grafted onto the cationic domain Otherwise, the ligand could be buried and thus unavailable for binding interactions Several heterobifunctional PEGs (abbreviated as X-PEG-Y) are commercially available in a 3.4-kDa size These X-PEG-Y molecules can be used to connect ligands to cationic domains An example of this type of synthesis is shown in Fig Trilactosyl aldehyde (100 mg, 0.067 mmol) is stirred in water (0.5 mL) under argon Amino-PEG3.4k-amino-t-BOC (151 mg, 0.04 mmol) and lithium hydroxide (1.7 mg, 0.04 mmol) dissolved in methanol (1 ml) are then added to the trigalactosyl aldehyde solution and stirred under argon at 25°C for 30 Two portions of sodium cyanoborohydride (6.2 mg, 0.1 mmol) are then added over a 24-h period Methyl iodide (568 mg, mmol) is added and the solution stirred for 24 h The solution is then evaporated to dryness, and trifluoroacetic acid (0.7 mL) in methylene chloride (0.6 mL) is added The solvents are again evaporated to dryness and the residue redissolved in a mixture of methanol and water (3 mL) The solution is adjusted to pH with 10 N sodium hydroxide Succinimidyl bromoacetate (118.5 mg, 0.5 mmol) is then added in acetonitrile (0.5 mL), and the mixture is stirred under argon at 25°C for h The bromoacetyl intermediate is eluted over a Sephadex G-25 column in 0.05 N acetic acid The macromolecular fractions are combined and evaporated in vacuo 10 Poly-L-lysine 26k (27.7 mg, 0.001 mmol) and lithium hydroxide (4.6 mg, 0.11 Poly-L-Lysine-Based Gene Delivery Systems Fig An example of stepwise grafting Lollo et al mmol) dissolved in methanol (1.5 mL) are added to the solution of iodoacetyl intermediate 11 The reaction mixture is sealed and incubated overnight at 37°C 12 The product is purified by SP Sepharose FF and Sephadex G-25 column chromatography 13 The ratio of triantennary galactose/PEG/PLL is determined by 1H NMR 3.3 1H NMR Spectroscopy Each polymer is first freeze-dried from D2O and redissolved in D2O for spectral analysis This procedure minimizes the HOD peak and gives superior spectra 1H NMR spectra are recorded on a high-resolution spectrometer (e.g., 300 MHz ARX-300 Bruker) Chemical shifts are expressed in parts per million and referenced to the HDO signal at 4.7 ppm The integration ratio of PEG signal (3.68 ppm) to Cα-H of poly-L-lysine (4.2 ppm) is used to determine the composition of the copolymer The number of Cα-H protons per PLL molecule is calculated from the MW and known structure For example, 10-kDa polylysine has 48 Cα-H The number of methylene protons (–CH2-) per PEG molecule is calculated from the MW and known structure For example, 5-kDa PEG has 454 methylene protons The number determined in step is divided by the number determined in step to yield the proton ratio expected for a 1:1 conjugation of PEG and PL The ratio computed in step is divided by the ratio computed in step to yield the average number of PEG grafts per PLL molecule 3.4 Plasmid DNA Preparation and purification of plasmid DNA is beyond the scope of this chapter, but a few salient points need to be made as to the use of plasmid DNA for polyplex formation and transfection studies These remarks assume that the plasmid was constructed properly, contains the proper elements, and is known to express at reasonable levels in transfection assays in vitro Plasmid DNA should be assayed by agarose gel electrophoresis with ethidium bromide staining to determine purity and relative amounts of linear and covalently closed circular forms including the super-coiled form For best results, plasmid DNA used in transfection studies should be ≥90% in the covalently closed circular form Plasmid DNA should be stored below 4°C in an appropriate buffer (e.g., 10 mM Tris(hydroxymethyl)aminomethane, mM EDTA, pH 8.0) DNA preparations must be tested for endotoxin levels using the limulus amebocyte lysate assay (Bio-Whittaker, Walkersville, MD) or other methods (19) Contamination should not exceed 10 endotoxin units per milligram of plasmid DNA Poly-L-Lysine-Based Gene Delivery Systems 3.5 Charge Ratio Determinations Charge ratios (+/-) can be determined by several methods, and it is recommended that at least two independent methods be used to characterize conjugates We recommend using a theoretical calculation based on composition combined with a fluorescence quenching assay 3.6 Calculation Based on Composition From the proton NMR data, calculate the expected molecular weight of the conjugate From the known composition of the conjugate, calculate the number of positive charges on each conjugate molecule Calculate the conjugate mass per positive charge (step 1/step 2) The mean mass per unit negative charge for plasmid DNA is 330 Conjugate mass per unit charge (step 3) divided by DNA mass per unit charge (330) is the theoretical mass ratio (R) to form a neutral polyplex To manufacture a polyplex at a given charge ratio, use the following equation: mass of conjugate = desired polyplex charge ratio × DNA mass × R 3.7 Fluorescence Quenching Assay The binding abilities of polycationic polymers were examined using an ethidium bromide-based quenching assay Solutions (1 mL) containing 2.5 µg/mL ethidium bromide and 10 µg/mL DNA (1:5 molar ratio, EtBr/DNA phosphate) are prepared Highly concentrated aqueous conjugate solutions (≥1 mg/mL) are used to minimize the effect of dilution after multiple additions Fluorescence reading is taken of the DNA solution prepared in step 1, using a fluorometer with excitation and emission wavelengths at 540 and 585 nm, respectively Aliquots of the conjugate solution prepared in step are added incrementally to the DNA solution, and fluorescence readings are taken after each addition Aliquots should be