Endosomolytic polymers

Duncan, R. Understanding the mechanism of action of 67. poly(amidoamine)s as endosomolytic polymers correlation of physicochemical and biological properties. Biomacromo-lecues 2004, 5, 1422-1427. 

Richardson, S. Ferruti, S. Duncan, R. Poly(amidoamine)s as potential endosomolytic polymers evaluation in vitro and body distribution in normal and tumor bearing animals. J. Drug Target. 1996, 6, 391-397. 

Ferruti P, Manzoni S, Richardson SCW, Duncan R, Pattrick NG, Mendichi R, Casolaro M. Amphoteric linear poly(amido-amine)s as endosomolytic polymers correlation between physico-chemical and biological properties. Macromolecules 2000 21 7793-7800. 

N. Lavignac, M. Lazenby, J. Franchini, P. Ferruti and R. Duncan, S5mthesis and preliminary evaluation of poly(amidoamine)-melittin conjugates as endosomolytic polymers and/or potential anticancer therapeutics, Int. J. Pharm., 300,102-112 (2005). 

Murthy 2003 cleavage of acetal-linked PEG endosomolytic polymer exposed endosomal escape of antisense oligonncleotides... 

Wong, S.C., Klein, J.J., Hamilton, H.L., Chu, Q., Frey, C.L., Trubetskoy, V.S., Hegge, J., Wakefield, D., Rozema, D.B., Lewis, D.L., 2012. Co-injection of a targeted, reversibly masked endosomolytic polymer dramatically improves the efficacy of cholesterol-conjugated small interfering RNAs in vivo. Nucleic Acid Ther. 22, 380-390. 

Other systems like electroporation have no lipids that might help in membrane sealing or fusion for direct transfer of the nucleic acid across membranes they have to generate transient pores, a process where efficiency is usually directly correlated with membrane destruction and cytotoxicity. Alternatively, like for the majority of polymer-based polyplexes, cellular uptake proceeds by clathrin- or caveolin-dependent and related endocytic pathways [152-156]. The polyplexes end up inside endosomes, and the membrane disruption happens in intracellular vesicles. It is noteworthy that several observed uptake processes may not be functional in delivery of bioactive material. Subsequent intracellular obstacles may render a specific pathway into a dead end [151, 154, 156]. With time, endosomal vesicles become slightly acidic (pH 5-6) and finally fuse with and mature into lysosomes. Therefore, polyplexes have to escape into the cytosol to avoid the nucleic acid-degrading lysosomal environment, and to deliver the therapeutic nucleic acid to the active site. Either the carrier polymer or a conjugated endosomolytic domain has to mediate this process [157], which involves local lipid membrane perturbation. Such a lipid membrane interaction could be a toxic event if occurring at the cell surface or mitochondrial membrane. Thus, polymers that show an endosome-specific membrane activity are favorable. 

First clinical human gene therapy trials with polyplexes were performed using cancer vaccines based on autologous patient tumor cells. These were modified ex vivo with interleukin-2 pDNA. To obtain high level transfection rates of patient s primary tumor cells, Tf-PLL/pDNA polyplexes linked with inactivated endosomolytic adenovirus particles were applied [221]. Polymer-based in vivo human gene transfer studies were performed with PEGylated PLL polyplexes, delivering CFTR pDNA to the airway epithelium of cystic fibrosis patients [222],... 

Meyer M, Dohmen C, Philipp A, Kiener D, Maiwald G, Scheu C, Ogris M, Wagner E (2009) Synthesis and biological evaluation of a bioresponsive and endosomolytic siRNA-polymer conjugate. Mol Pharmaceutics 6 752-762... 

Once internalized, the essential step for the polyplex is to escape rapidly the endosomal vesicle in order to release the nucleic acid in the cytosol and prevent its lysosomal degradation. As the endosomal and lysosomal pH presents values between 4.5 and 6.5 and therefore differs from the neutral pH of 7.4 in other biological compartments [58], some polycations containing protonable residues like PEI facilitate this step by the proton sponge effect [59, 60]. As not all cationic polymers display this attribute, another effective method for enhanced endosomal polyplex release is incorporation of specific endosomal membrane disrupting or pore-forming domains, such as lytic lipid moieties or endosomolytic peptides. 

Fig. 2 Masked endosomolytic agents for pH-triggered endosomal escape. The polymers designed by Meyer et al. [69] (a) and Rozema et al. [71] (b) contain endosomolytic compounds whose lytic potential is activated by endosomal cleavage of masking groups coupled by acid-sensitive linkages. Furthermore, disulfide bonds are embedded which release the nucleic acid after endosomal escape by cleavage in the reducing cytosolic environment...

NMR measurements were in good agreement with those obtained from the SANS studies. These studies support the proposed endosomolytic activity of the PAA polymers. 

Of the many nonlipidic polycation gene delivery systems developed in the past decades, poly(L-lysine) (PLL) was the hrst polycation used for nonviral gene delivery [34]. Among a vast number of other positively charged polymers, polyethyleni-mine (PEI) has been widely used for nonviral transfection in vitro and in vivo and has an advantage over other polycations in that it combines strong DNA condensation capacity with an intrinsic endosomolytic activity [35-38]. 

This was the basis for the development of a second generation DPC, named ARC-520, which is currently in phase II clinical trial for the treatment of hepatitis B (Table 14.2). In ARC-520, the previous endosomolytic PBAVE polymer was replaced with a melittin-like peptide (MLP) with a well-defined structure. In addition, there is no chemical link between the siRNA and the polymer. Thus, ARC-520 consists of two individual components cholesterol-siRNA conjugates and hepatocyte-targeting membrane-active peptides linked to the MLP polymer. 

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