STRATEGIES AND TECHNOLOGIES TO OVERCOME BARRIERS

Chemical Capsid Modifications

Compared to genetic modifications, chemical modifications have the advantage that they can attach molecules derived from different substance classes to the capsid surface of the virions. This includes molecules that cannot be encoded genetically (e.g., carbohydrates (Espenlaub et al. 2008)) or those that are fully synthetic. In particular, inert synthetic polymers that act as steric shields have been attached to the surface of Ad vectors.

Chemical Capsid Modifications with Synthetic Polymers

Polymer shielding uses (synthetic) polymers, which are hydrophilic and bulky, to create a steric shield around the capsid surface. Typically, the synthetic polymers are covalently attached to the virus surface by chemical reactions that maintain the structural integrity of the capsid. The size and the chemical nature of the polymer

Schematic illustrations of polymer-coated “shielded” Ad vectors

FIGURE 4.2 Schematic illustrations of polymer-coated “shielded” Ad vectors. (a) Illustration of an Ad covalently coated with a synthetic polymer that reacted with one end with the capsid surface (“semitelechelic polymer”). The other end protrudes from the capsid surface and is freely movable. This is typical for PEGylated Ads. (b) Illustration of an Ad covalently coated with a synthetic polymer that reacted with the capsid surface at multiple reactive sites per polymer molecule. This is typical for HPMAylated Ads. The protruding distal ends of semitelechelic PEG molecules and unreacted groups on HPMA molecules can be used to couple ligands for targeting to the polymer shield. (c) Semitelechelic PEG molecule with one reactive end bearing an NHS ester that is reactive towards amine groups which are abundant on the surface of Ad capsids. (d) Heterobifunctional PEG molecule that is suitable for coupling of ligands to the capsid surface. (e) pHPMA molecules for multivalent coupling to the capsid surface.

as well as the mode of covalent attachment to the capsid surface determine to which degree the polymers sterically prevent interactions of the virus surface with the environment. Figure 4.2a,b shows a schematic illustration of polymer-conjugated Ad.

One of the most important advantages of chemical capsid modifications compared to genetic approaches is that the chemical reaction to attach the polymer can be performed after vector production and purification. Thus, there is no interference with vector production and conventional and well-characterized producer cells can be used and stocks with high vector titers can easily be obtained using standard methods. In addition, chemical capsid modification can be performed in a way that simultaneously modifies up to several thousands of amino acid residues on the capsid surface. Such extensive alterations of the capsid surface would likely be impossible to achieve by genetic modifications.

It has to be noted though—in particular in the context of conditionally replication-competent vectors—that the surface modifications performed by chemistry after vector production will not be passed onto progeny virions (in contrast of course to genetic modifications). However, chemical and genetic modifications as well as the use of different Ad types do not exclude each other and a combination of the approaches appears promising (Nguyen et al. 2016).

Two types of synthetic polymers have been used for covalent modification of Ad gene transfer vectors. The first is based on PEG, the second is based on poly-N- hydroxypropylmethacrylamide (pHMPA).

 
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