Endosomolytic activity

Meyer M, Zintchenko A, Ogris M, Wagner E (2007) A dimethylmaleic acid-melittin-polylysine conjugate with reduced toxicity, pH-triggered endosomolytic activity and enhanced gene transfer potential. J Gene Med 9 797-805... 

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

KALA pH-dependent endosomolytic activity by adopting an a-helical conformation WEAKLAKALAKALAKHLAKALA KALKACEA [63, 64]... 

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]. 

Adenoviruses consist of a large double-stranded DNA genome (about 36 kilobase pairs long) packaged within a nonenveloped icosahedral capsid that is primarily composed of three virus-encoded proteins (hexon, pen ton base, and fiber proteins) [39]. The fiber proteins protrude fi om the surface of the virus and mediate its attachment to target cells via a high affinity interaction with the cellular receptor CAR (coxsackievirus and adenovirus receptor) [40]. The virus is then internahzed into endosomal vesicles via specific interactions between the penton base proteins and ofy integrins [41]. Adenoviruses escape these vesicles by an acid-induced endosomolytic activity and are transported to the nucleus, into which they enter via pores in the nuclear membrane [42]. 

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. 

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...

As PAA exist in the form of a salt, it is important to determine, prior to use as endosomolytic agents, which, if any salt, exhibits the most appropriate pH-membrane perturbation properties. A SANS study was therefore initiated to investigate systematically the effects of pH and counterion on the molecular structure (as assessed by Rg) of the PAA, ISA 23 when dispersed in D2O, and to correlate molecular structure with its hemolytic activity. Fig. 10 shows the normalized... 

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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