the hexon, fiber, or knob, with similar proteins from other virus
serotypes, species, or families. Wu et al. demonstrated that
exchanging Ad-5 and Ad-3 hexon proteins produced a virus capable of evading neutralizing antibodies that recognize the Ad-5
vector [8]. Similarly, replacing the Ad-5 knob with that of Ad-3
was shown to alter virus tropism and enhance binding and entry
into melanoma cells [9].
Chemical modification of the virus includes covalently or noncovalently complexing polymers to the surface of the virus. For
example, the surface of Ad-5 has been chemical modified using
polymers such as polyethylene glycol (PEG), poly-N-(2-hydroxypropyl) methacrylamide (pHPMA), poly(ethylenimine) (PEI), poly
(L-lysine) (PLL), and chitosan [10–14]. PEGylating adenovirus
has been shown to prolong systemic circulation, reduce liver transduction, and reduce the innate immune response [15, 16]. PEGylation, however, reduces infection efficiency of Ad through steric
hindrance [17, 18].
An approach that our group has taken, which has shown success
in addressing some of the aforementioned drawbacks, is to PEGylate the surface of the virus and subsequently attach peptides to the
virus to improve uptake or target desired cells (Fig. 1)
[19, 20]. The protocol outlined here describes our approach for
PEGylating adenovirus and attaching cell-penetrating peptides
(CPPs). PEGylation reduces the immunogenicity of the virus and
also improves its resistance to inactivation by neutralizing antibodies. PEGylation, however, typically inhibits uptake of particles and
so the addition of CPPs is necessary to restore infectivity or impart
infectivity on CAR-negative cells.
The approach described here can be extended to a variety of
polymers and distal moieties (antibodies, peptides, aptamers, etc.).
The general approach is to use a heterobifunctional PEG,
maleimide-PEG-hydroxysuccinimide ester, to PEGylate the virus
though a reaction with the terminal amine group within the capsid
lysine residues. The thiol-reactive maleimide end of the polymer is
subsequently reacted with a cysteine residue added to the end of the
CPP. The details of this approach are presented below.
2 Materials
Work with adenovirus must be carried out in a class II biological
safety cabinet. All pipette tips, microcentrifuge tubes, and 15 ml
conical centrifuge tubes should be sterile. Lab personnel should
wear proper PPE including a laboratory coat, safety glasses, and
gloves. Virus-containing material should be autoclaved or disinfected and disposed of as biohazardous waste.
358
Yasmine Gabal and Joshua D. Ramsey
serotypes, species, or families. Wu et al. demonstrated that
exchanging Ad-5 and Ad-3 hexon proteins produced a virus capable of evading neutralizing antibodies that recognize the Ad-5
vector [8]. Similarly, replacing the Ad-5 knob with that of Ad-3
was shown to alter virus tropism and enhance binding and entry
into melanoma cells [9].
Chemical modification of the virus includes covalently or noncovalently complexing polymers to the surface of the virus. For
example, the surface of Ad-5 has been chemical modified using
polymers such as polyethylene glycol (PEG), poly-N-(2-hydroxypropyl) methacrylamide (pHPMA), poly(ethylenimine) (PEI), poly
(L-lysine) (PLL), and chitosan [10–14]. PEGylating adenovirus
has been shown to prolong systemic circulation, reduce liver transduction, and reduce the innate immune response [15, 16]. PEGylation, however, reduces infection efficiency of Ad through steric
hindrance [17, 18].
An approach that our group has taken, which has shown success
in addressing some of the aforementioned drawbacks, is to PEGylate the surface of the virus and subsequently attach peptides to the
virus to improve uptake or target desired cells (Fig. 1)
[19, 20]. The protocol outlined here describes our approach for
PEGylating adenovirus and attaching cell-penetrating peptides
(CPPs). PEGylation reduces the immunogenicity of the virus and
also improves its resistance to inactivation by neutralizing antibodies. PEGylation, however, typically inhibits uptake of particles and
so the addition of CPPs is necessary to restore infectivity or impart
infectivity on CAR-negative cells.
The approach described here can be extended to a variety of
polymers and distal moieties (antibodies, peptides, aptamers, etc.).
The general approach is to use a heterobifunctional PEG,
maleimide-PEG-hydroxysuccinimide ester, to PEGylate the virus
though a reaction with the terminal amine group within the capsid
lysine residues. The thiol-reactive maleimide end of the polymer is
subsequently reacted with a cysteine residue added to the end of the
CPP. The details of this approach are presented below.
2 Materials
Work with adenovirus must be carried out in a class II biological
safety cabinet. All pipette tips, microcentrifuge tubes, and 15 ml
conical centrifuge tubes should be sterile. Lab personnel should
wear proper PPE including a laboratory coat, safety glasses, and
gloves. Virus-containing material should be autoclaved or disinfected and disposed of as biohazardous waste.
358
Yasmine Gabal and Joshua D. Ramsey