NOTE: In the following, a protocol for geneti-chemical PEGylation of an Ad vector is described to detail. To enable specific coupling of the PEG moiety, an Ad5 vector was beforehand genetically modified by introducing a cysteine residue into the hexon protein at the hypervariable loop 5 as described in a previous publication36, and a maleimide-activated PEG compound is used as coupling compound.
1. Preparation of Buffers for Vector Purification by CsCL Step Gradients
...

<ol>
	<li>Benko, M., Harrach, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+evolution+of+adenoviruses.">Molecular evolution of adenoviruses.</a> Current Topics in Microbiology and Immunology. , 3-35 (2003).</li><li>Davison, A. J., Benko, M., Harrach, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+content+and+evolution+of+adenoviruses.">Genetic content and evolution of adenoviruses.</a> Journal of Genetic Virology. 84, 2895-2908 (2003).</li><li>Rowe, W. P., Huebner, R. J., Gilmore, L. K., Parrott, R. H., Ward, T. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+reconstruction+reveals+the+complex+molecular+organization+of+adenovirus.">Image reconstruction reveals the complex molecular organization of adenovirus.</a> Cell. 67, 145-154 (1991).</li><li>Meier, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus+triggers+macropinocytosis+and+endosomal+leakage+together+with+its+clathrin-mediated+uptake.">Adenovirus triggers macropinocytosis and endosomal leakage together with its clathrin-mediated uptake.</a> Journal of Cellular Biology. 158, 1119-1131 (2002).</li><li>Xu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coagulation+factor+X+shields+adenovirus+type+5+from+attack+by+natural+antibodies+and+complement.">Coagulation factor X shields adenovirus type 5 from attack by natural antibodies and complement.</a> Nature Medicine. 19, 452-457 (2013).</li><li>Qiu, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+natural+IgM+concentration+on+gene+therapy+with+adenovirus+type+5+vectors.">Impact of natural IgM concentration on gene therapy with adenovirus type 5 vectors.</a> Journal of Virology. 89, 3412-3416 (2015).</li><li>Alemany, R., Suzuki, K., Curiel, D. 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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+efficient+clearance+of+blood-borne+virus+by+liver+sinusoidal+endothelium.">Rapid and efficient clearance of blood-borne virus by liver sinusoidal endothelium.</a> PLoS Pathogen. 7, e1002281 (2011).</li><li>Cichon, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Titer+determination+of+Ad5+in+blood:+a+cautionary+note.">Titer determination of Ad5 in blood: a cautionary note.</a> Gene Therapy. 10, 1012-1017 (2003).</li><li>Carlisle, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+erythrocytes+bind+and+inactivate+type+5+adenovirus+by+presenting+Coxsackie+virus-adenovirus+receptor+and+complement+receptor+1.">Human erythrocytes bind and inactivate type 5 adenovirus by presenting Coxsackie virus-adenovirus receptor and complement receptor 1.</a> Blood. 113, 1909-1918 (2009).</li><li>Waddington, S. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus+serotype+5+hexon+mediates+liver+gene+transfer.">Adenovirus serotype 5 hexon mediates liver gene transfer.</a> Cell. 132, 397-409 (2008).</li><li>Kalyuzhniy, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus+serotype+5+hexon+is+critical+for+virus+infection+of+hepatocytes+in+vivo.">Adenovirus serotype 5 hexon is critical for virus infection of hepatocytes in vivo.</a> Proceedings of the National Academy of Sciences USA. 105, 5483-5488 (2008).</li><li>Vigant, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substitution+of+hexon+hypervariable+region+5+of+adenovirus+serotype+5+abrogates+blood+factor+binding+and+limits+gene+transfer+to+liver.+Molecular+Therapy.">Substitution of hexon hypervariable region 5 of adenovirus serotype 5 abrogates blood factor binding and limits gene transfer to liver. Molecular Therapy.</a> The Journal of the American Society of Gene Therapy. 16, 1474-1480 (2008).</li><li>Jonsson, M. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coagulation+factors+IX+and+X+enhance+binding+and+infection+of+adenovirus+types+5+and+31+in+human+epithelial+cells.">Coagulation factors IX and X enhance binding and infection of adenovirus types 5 and 31 in human epithelial cells.</a> Journal of Virology. 83, 3816-3825 (2009).</li><li>Bradshaw, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirements+for+receptor+engagement+during+infection+by+adenovirus+complexed+with+blood+coagulation+factor+X.">Requirements for receptor engagement during infection by adenovirus complexed with blood coagulation factor X.</a> PLoS Pathogen. 6, e1001142 (2010).</li><li>Duffy, M. R., Bradshaw, A. C., Parker, A. L., McVey, J. H., Baker, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cluster+of+basic+amino+acids+in+the+factor+X+serine+protease+mediates+surface+attachment+of+adenovirus/FX+complexes.">A cluster of basic amino acids in the factor X serine protease mediates surface attachment of adenovirus/FX complexes.</a> Journal of Virology. 85, 10914-10919 (2011).</li><li>Xu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coagulation+factor+X+shields+adenovirus+type+5+from+attack+by+natural+antibodies+and+complement.">Coagulation factor X shields adenovirus type 5 from attack by natural antibodies and complement.</a> Nature Medicine. 19, 452-457 (2013).</li><li>Prill, J. -. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modifications+of+adenovirus+hexon+allow+for+either+hepatocyte+detargeting+or+targeting+with+potential+evasion+from+Kupffer+cells.+Molecular+Therapy.">Modifications of adenovirus hexon allow for either hepatocyte detargeting or targeting with potential evasion from Kupffer cells. Molecular Therapy.</a> The Journal of the American Society of Gene Therapy. 19, 83-92 (2011).</li><li>Zaiss, A. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanomedicine:+clinical+applications+of+polyethylene+glycol+conjugated+proteins+and+drugs.">Nanomedicine: clinical applications of polyethylene glycol conjugated proteins and drugs.</a> Clin. Pharmacokinet. 45, 965-988 (2006).</li><li>O'Riordan, C. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PEGylation+of+adenovirus+with+retention+of+infectivity+and+protection+from+neutralizing+antibody+in+vitro+and+in+vivo.">PEGylation of adenovirus with retention of infectivity and protection from neutralizing antibody in vitro and in vivo.</a> Human Gene Therapy. 10, 1349-1358 (1999).</li><li>Subr, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coating+of+adenovirus+type+5+with+polymers+containing+quaternary+amines+prevents+binding+to+blood+components.">Coating of adenovirus type 5 with polymers containing quaternary amines prevents binding to blood components.</a> Journal of Controlled Release. 135, 152-158 (2009).</li><li>Kreppel, F., Gackowski, J., Schmidt, E., Kochanek, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+genetic+and+chemical+capsid+modifications+enable+flexible+and+efficient+de-+and+retargeting+of+adenovirus+vectors.+Molecular+Therapy.">Combined genetic and chemical capsid modifications enable flexible and efficient de- and retargeting of adenovirus vectors. Molecular Therapy.</a> The Journal of the American Society of Gene Therapy. 12, 107-117 (2005).</li><li>Corjon, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+of+adenovirus+vectors+to+the+LRP+receptor+family+with+the+high-affinity+ligand+RAP+via+combined+genetic+and+chemical+modification+of+the+pIX+capsomere.">Targeting of adenovirus vectors to the LRP receptor family with the high-affinity ligand RAP via combined genetic and chemical modification of the pIX capsomere.</a> Molecular Therapy: The Journal of the American Society of Gene Therapy. 16 (11), 1813-1824 (2008).</li><li>Prill, J. -. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modifications+of+adenovirus+hexon+allow+for+either+hepatocyte+detargeting+or+targeting+with+potential+evasion+from+Kupffer+cells.+Molecular+Therapy.">Modifications of adenovirus hexon allow for either hepatocyte detargeting or targeting with potential evasion from Kupffer cells. Molecular Therapy.</a> The Journal of the American Society of Gene Therapy. 19, 83-92 (2011).</li><li>Krutzke, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substitution+of+blood+coagulation+factor+X-binding+to+Ad5+by+position-specific+PEGylation:+Preventing+vector+clearance+and+preserving+infectivity.">Substitution of blood coagulation factor X-binding to Ad5 by position-specific PEGylation: Preventing vector clearance and preserving infectivity.</a> Journal of Controlled Release. 235, 379-392 (2016).</li><li>Prill, J. -. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traceless+bioresponsive+shielding+of+adenovirus+hexon+with+HPMA+copolymers+maintains+transduction+capacity+in+vitro+and+in+vivo.">Traceless bioresponsive shielding of adenovirus hexon with HPMA copolymers maintains transduction capacity in vitro and in vivo.</a> PloS One. 9, e82716 (2014).</li><li>Kratzer, R. F., Kreppel, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production,+Purification,+and+Titration+of+First-Generation+Adenovirus+Vectors.">Production, Purification, and Titration of First-Generation Adenovirus Vectors.</a> Methods in Molecular Biology. , 377-388 (2017).</li><li>Blum, H., Beier, H., Gross, H. 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The efficiency by which the genetically introduced cysteines can be chemically modified is typically 80-99%, and certain variables influence this efficiency. First, it is paramount that the genetically introduced cysteines do not undergo premature oxidation. While being well-protected in the reducing environment of the producer cells, it is mandatory to provide a non-oxidative environment after releasing vector particles from the producer cells and during chemical modification. To this end, reducing reagents can be used ...

Adenoviruses (Ad), members of the family Adenoviridae, are non-enveloped DNA viruses of which more than 70 types so far have been identified (http://hadvwg.gmu.edu). Depending on hemaglutination properties, genome structure, and sequencing results, the 70 Ad types can be divided into seven species (human adenoviruses A to G)1,2. The human Ad genome is 38 kb in size and encapsulated by an icosahedral nucleocapsid3. Due to their abundance, the capsid protein hexon, penton base, and fiber are all referred to as major capsid proteins. The most abundant and largest capsid protein hexon forms 20 capsid facets, each consisting of 12 hexon homotrimers4,5. Penton, located on each icosahedral edge (vertex), consists of pentamers of a penton base and represents the base for the vertex spike that is built of glycosylated fiber trimers5,6. The native Ad cell entry is basically composed of two major steps. First, the fiber knob binds to the primary receptor. In Ad types from species A, C, E, and F, this is the coxsackie and adenovirus receptor (CAR). This interaction brings the virion into spatial proximity of the cell surface, thereby facilitating interactions between cellular integrins and RGD-motif in the penton base and consequently inducing cellular responses. Second, changes in the cytoskeleton lead to internalization of the virion and transport to the endosome7. Upon partial disassembly in the endosome, the virion is released to the cytoplasm und ultimately travels to the nucleus for replication.
While Ad can be delivered locally (e.g., for genetic vaccination), systemic delivery through the bloodstream as required for onco-virotherapy faces several barriers. While circulating in the bloodstream, injected virions encounter the defense system of the host&#39;s immune system, leading to fast neutralization of the virus-based vectors and rendering Ad-based vectors extremely inefficient in systemic applications. Furthermore, the natural hepatotropism of Ad interferes with systemic delivery and must be resolved to redirect Ad to its new target cells.
Germline-encoded natural IgM antibodies of the innate immune system rapidly recognize and bind highly repetitive structures on the surface of the virion8,9. These immune complexes then activate the classical and non-classical pathways of the complement system, leading to fast complement-mediated neutralization of a large portion of the virions8. A second pathway resulting in major removal of Ad virions is mediated by macrophages10&#160;and associated with acute toxic and haemodynamic side effects11,12. In the case of Ad in particular, Kupffer cells residing in the liver bind to and phagocytically take up the Ad virions via specific scavenger receptors, thereby eliminating them from the blood13,14,15. Specific scavenger receptors have also been identified on liver sinusoidal endothelial cells (LSE cells)16,and LSE cells also seem to contribute to vector elimination17, but to what extent still needs clarification. Furthermore, some Ad types and their&#160;derived vectors are efficiently sequestered by human erythrocytes18&#160;to which they bind via CAR or the complement receptor CR119.&#160;Of note, this sequestering mechanism cannot be studied in the mouse model system as in contrast to human erythrocytes, mouse erythrocytes do not express CAR.
Specific anti-Ad antibodies generated by the adaptive immune system after exposure to Ad either due to previous infections with Ad or after the first delivery in systemic applications raise a further barrier to effective use of Ad vectors, and they should be evaded in efficient systemic delivery.
Finally, the strong hepatotropism of some Ad types (including Ad5) severely hinders application of Ad in systemic therapy. This tropism resulting in hepatocyte transduction is due to the high affinity of the Ad virion to blood coagulation factor X (FX), mediated by the interaction of FX with the Ad hexon protein20,21,22. FX bridges the virion to heparin sulfate glycans (HSPGs) on the surface of hepatocytes20,23,24,25. A crucial factor for this interaction seems to be the specific extent of N- and O-sulfation of the HSPGs in liver cells24, which is distinct from HSPGs on other cell types. In addition to this FX-mediated pathway, recent studies suggest further pathways not yet identified that result in Ad transduction of hepatocytes26,27,28.
Recently, it has been shown that FX is not only involved in hepatocyte transduction of Ad, but also by binding, the virion shields the virus particle against neutralization by the complement system26. Reduction of hepatocyte transduction by preventing FX binding, therefore, would create the unwanted side effect of increasing Ad neutralization via the innate immune system.
A profound knowledge of the complex interactions between vectors and host organisms is therefore necessary to develop more efficient vectors for systemic applications that circumvent the obstacles imposed by the host&#39;s organism.
One strategy that has been originally used for therapeutic proteins has been adapted for Ad vectors to at least partially overcome the above described barriers. Antigenicity and immunogenicity of therapeutic protein compounds could be reduced by coupling to polyethylene glycol (PEG)29,30. Hence, the covalent coupling of polymers such as PEG or poly[N-(2-hydroxypropyl)methacrylamid] (pHPMA) to the capsid surface shields the vector from unwanted vector-host interactions. Commonly, polymer coupling targets &#949;-amine groups from lysine side residues that are randomly distributed on the capsid surface. Vector particles in solution are, due to the hydrophilic nature of the attached polymers, surrounded by a stable water shell that reduces the risk of immune cell recognition or enzymatic degradation. Moreover, PEGylated Ad vectors were shown to evade neutralization by anti-hexon antibodies in vitro and in pre-immunized mice in vivo31. In contrast to genetic capsid modifications, chemical coupling of polymers is performed after production and purification, allowing not only for the use of conventional producer cells and production of high titer vector stocks, but also for simultaneous modification of thousands of amino acids on the capsid surface. However, amine-directed shielding occurs randomly throughout the whole capsid surface, resulting in high heterogeneities and not allowing for modification of specific capsomers. Furthermore, the large polymer moieties required for beneficial effects impair virus bioactivity32.
To overcome these limitations, Kreppel et al.33 introduced a geneti-chemical concept for vector re- and de-targeting. Cysteines were genetically introduced into the virus capsid at solvent-exposed positions like fiber HI-loop33, protein IX34, and hexon35,36. Although not naturally-occurring, cysteine-bearing Ad vectors can be produced at high titers in normal producer cells. Importantly, insertion of cysteines in certain capsomers and in different positions within a single capsomer allows for highly specific modifications of thiol group-reactive moieties. This geneti-chemical approach has been shown to overcome numerous obstacles in Ad vector design. The combination of amine-based PEGylation for detargeting and thiol-based coupling of transferrin to the fiber knob HI-loop has been proven to successfully retarget modified Ad vectors to CAR-deficient cells33. Since hexon is involved in most undesired interactions (neutralizing antibodies, blood coagulation factor FX), thiol-based modification strategies were also applied to hexon. Coupling small PEG moieties to HVR5 of hexon prevented Ad vector particles to transduce SKOV-3 cells in the presence of FX, whereas large PEG moieties increased hepatocyte transduction14,35. Ad vector particles carrying mutations in the fiber knob inhibiting CAR binding and in HVR7 inhibiting binding of FX (and bearing inserted cysteines in HVR1 for position-specific PEGylation) were shown to evade antibody- and complement-mediated neutralization, as well as scavenger receptor-mediated uptake without loss of infectivity. Interestingly, despite a lack of the natural FX shield, PEGylation again improved transduction of hepatocytes as a function of PEG size36. However, it was shown that covalent shielding does have an impact on intracellular trafficking processes. Prill et al. compared irreversible versus bioresponsive shields based on pHPMA and demonstrated that neither the mode of shielding nor co-polymer charge had an impact on cell entry but did affect particle trafficking to the nucleus. Employing a bioresponsive shield with positively charged pHPMA co-polymers allowed for particle trafficking to the nucleus, maintaining the high transduction efficiencies of Ad vectors in vitro and in vivo37.
In summary, these data indicate that, even under the assumption that all vector-host-interactions were known and considered, excessive capsid surface modifications are necessary to overcome the hurdles associated with systemic vector delivery.
Here we provide a protocol to perform site-specific chemical modifications of adenovirus vector capsids for shielding and/or retargeting of adenovirus vector particles and adenovirus-based oncolytic viruses. The concept of this technology is outlined in Figure 1. It allows the shielding of certain capsid regions from unwanted interactions by covalent attachment of synthetic polymers. At the same, it also provides a means to attach ligands and combine shielding and targeting. Using simple chemistry, experimenters will be able to covalently modify adenovirus vector surface with a wide variety of molecules including peptides/proteins, carbohydrates, lipids, and other small molecules. Furthermore, the protocol provides a general concept for the chemical modification of biologically active virus-derived vectors under maintenance of their biological integrity and activity.

<table>
	<tbody>
		<tr>
			<td>Vector purification and chemical modification</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Argon gas</td>
			<td>Air liquide</td>
			<td>local gas dealer</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid Nitrogen</td>
			<td>Air liquide</td>
			<td>local gas dealer</td>
			<td></td>
		</tr>
		<tr>
			<td>500 mL centrifuge tubes</td>
			<td>Corning</td>
			<td>431123</td>
			<td></td>
		</tr>
		<tr>
			<td>Stericup Express Plus 0.22 &#181;m</td>
			<td>Millipore</td>
			<td>SCGPU02RE</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris(2-carboxyethyl) phosphine (TCEP)</td>
			<td>Sigma-Aldrich</td>
			<td>C4706-10g</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL (3mL) Norm Ject (syringes)</td>
			<td>Henke Sass Wolf</td>
			<td>4020.000V0</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine-Ject needles for single use (yellow 0.9 x 40 mm)</td>
			<td>Henke Sass Wolf</td>
			<td>4710009040</td>
			<td></td>
		</tr>
		<tr>
			<td>Caesium chloride 99.999% Ultra Quality</td>
			<td>Roth</td>
			<td>8627.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Silica gel beads</td>
			<td>Applichem</td>
			<td>A4569.2500</td>
			<td></td>
		</tr>
		<tr>
			<td>Methoxypolyethylene glycol maleimide - 750 (PEG mal-750)</td>
			<td>Iris Biotech</td>
			<td></td>
			<td>store in silica gel beads at -80 &#176;C</td>
		</tr>
		<tr>
			<td>13.2 mL Ultra Clear Ultracentrifuge Tubes</td>
			<td>Beckman Coulter</td>
			<td>344059</td>
			<td>only open in hood</td>
		</tr>
		<tr>
			<td>PD-10 size exclusion chromatography column</td>
			<td>GE Healthcare</td>
			<td>17-0851-01</td>
			<td>store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>AppliChem</td>
			<td>A1069.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS Ultrapure</td>
			<td>AppliChem</td>
			<td>A1112,0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>AppliChem</td>
			<td>A1123.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Material for cell-culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>PAN Biotech</td>
			<td>P04-36500</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>PAN Biotech</td>
			<td>P04-03590</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/EDTA</td>
			<td>PAN Biotech</td>
			<td>P10-0231SP</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS Good</td>
			<td>PAN Biotech</td>
			<td>P40-37500</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>PAN Biotech</td>
			<td>P06-07100</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosphere Filter Tips (various sizes)</td>
			<td>Sarstedt</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Serological Pipettes (various sizes)</td>
			<td>Sarstedt</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>reaction tubes (various sizes)</td>
			<td>Sarstedt</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TC plates 15cm</td>
			<td>Sarstedt</td>
			<td>83.3903</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Material for silver staining protocol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>J.T.Baker</td>
			<td>8045</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>AppliChem</td>
			<td>1613,2500PE</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>AppliChem</td>
			<td>A0820,2500PE</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde 37%</td>
			<td>AppliChem</td>
			<td>A0877,0250</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>AppliChem</td>
			<td>A1613,2500PE</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium thiosulfate</td>
			<td>AppliChem</td>
			<td>1,418,791,210</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver nitrate</td>
			<td>AppliChem</td>
			<td>A3944.0025</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium carbonate</td>
			<td>AppliChem</td>
			<td>A3900,0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Special Lab Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Desiccator</td>
			<td>Nalgene</td>
			<td>5311-0250</td>
			<td></td>
		</tr>
		<tr>
			<td>Megafuge 40</td>
			<td>Heraeus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Roter for Megafuge TX750 + Adapter andLlids for 500 mL tubes</td>
			<td>Heraeus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Conventional</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge e.g. Optima XPN-80</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>suitable Ultrazentrifuge Rotor e.g. SW41</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pH -Meter</td>
			<td>Conventional</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stand with clamps</td>
			<td>Conventional</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Goose neck lamp</td>
			<td>Conventional</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Over-head rotor</td>
			<td>Conventional</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thermal Block</td>
			<td>Conventional</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Photometer (OD 260)</td>
			<td>Conventional</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

combined genetic and chemical capsid modifications of adenovirus-based gene transfer vectors for shielding and targeting

Adenovirus vectors are potent tools for genetic vaccination and oncolytic virotherapy. However, they are prone to multiple undesired vector-host interactions, especially after in vivo delivery. It is a consensus that the limitations imposed by undesired vector-host interactions can only be overcome if defined modifications of the vector surface are performed. These modifications include shielding of the particles from unwanted interactions and targeting by the introduction of new ligands. The goal of the protocol presented here is to enable the reader to generate shielded and, if desired, retargeted human adenovirus gene transfer vectors or oncolytic viruses. The protocol will enable researchers to modify the surface of adenovirus vector capsids by specific chemical attachment of synthetic polymers, carbohydrates, lipids, or other biological or chemical moieties. It describes the cutting-edge technology of combined genetic and chemical capsid modifications, which have been shown to facilitate the understanding and overcoming of barriers for in vivo delivery of adenovirus vectors. A detailed and commented description of the crucial steps for performing specific chemical reactions with biologically active viruses or virus-derived vectors is provided. The technology described in the protocol is based on the genetic introduction of (naturally absent) cysteine residues into solvent-exposed loops of adenovirus-derived vectors. These cysteine residues provide a specific chemical reactivity that can, after production of the vectors to high titers, be exploited for highly specific and efficient covalent chemical coupling of molecules from a wide variety of substance classes to the vector particles. Importantly, this protocol can easily be adapted to perform a broad variety of different (non-thiol-based) chemical modifications of adenovirus vector capsids. Finally, it is likely that non-enveloped virus-based gene transfer vectors other than adenovirus can be modified from the basis of this protocol.

Figure 2 shows examples of the cytopathic effect (CPE) on 293 (HEK 293) cells that indicates successful vector production. Cells should show morphology (Figure 2C) 40-48 hours after inoculation with the virus vector. The right timepoint for harvesting is crucial for not losing virus particles by cell lysis and preventing oxidation of the genetically introduced thiol groups. If vector particles are released into the medium by cell...

Watch this Scientific Journal Video about Combined Genetic and Chemical Capsid Modifications of Adenovirus-Based Gene Transfer Vectors for Shielding and Targeting at JoVE.com

Combined Genetic and Chemical Capsid Modifications of Adenovirus-Based Gene Transfer Vectors for Shielding and Targeting

The protocol described here enables researchers to specifically modify adenovirus capsids at selected sites by simple chemistry. Shielded adenovirus vectors particles and retargeted gene transfer vectors can be generated, and vector host interactions can be studied.

Adenovirus vectors are potent tools for genetic vaccination and oncolytic virotherapy. However, they are prone to multiple undesired vector-host ...

This method can help to answer key questions in the field of Gene Therapy, Oncolysis and Genetic Vaccination. It facilitates understanding and modulating of vector hosts interactions of adenovirus gene transfer vectors, the most frequently used vector type in clinical trials up to date. The main advantage of this technique is that adenovirus capsulates can be specifically modified in a highly defined manner at selected positions and to variable extent by using simple chemistry.The implications of this technique extends towards therapy because vector host modification cannot only be analyzed but also modulated in a patient oriented way. Though this method provides insight into specific vector host interactions, it may also be used for labeling and tracking of virus particles in living organisms, such as mice. Generally, individuals new to this method will struggle with the unique combination of Virology and Organic Cchemistry that requires practical knowledge from both fields.Special demonstration of this technique is critical because here methods from Organic Chemistry meet methods from Cell Biology and Virology, including field-specific handling tricks. To begin this procedure prepare all buffers as outlined in the text protocol. Weigh one milliliter of each gradient buffer to check its density.After sterilizing all the buffers, sparge the buffers containing TCEP with argon gas for about 30 seconds to prevent oxidation of the TCEP. Store the vials containing the solid powdered coupling moiety in larger tubes, each filled with a cushion of silica gel beads, to prevent the build up of condensing water when thawing the vials. Place these tubes into a desiccator.Remove the air in the desiccator and replace it with argon gas. Then close each tube tightly and transfer it into a bigger container with silica gel beads. Once all the tubes have been transferred, close the container.Store the container at 80 degrees Celsius until ready to use. When ready to thaw, place the container into a layer of silica beads in a desiccator. Allow the container to slowly warm to room temperature and then open the container.When cells reach the optimal cytopathic affect, collect them by scraping the plates and transferring the supernatant to centrifuge tubes. Spin the cell suspension at 400 times G for 10 minutes. Next, re-suspend the cell pellet in four milliliters of Ad buffer containing 10 millimolar TCEP and transfer the resulting solution to a sterile 50 milliliter tube.Freeze the solution in liquid nitrogen, and then thaw it in a water bath at 37 degrees Celsius. Repeat this freeze thaw cycle three times to rescue the vector. Centrifuge at 5000 G and four degrees Celsius for ten minutes.While centrifuging set out two ultra centrifuge tubes to begin preparing two equal Cesium chloride step gradients. Add three milliliters of the lower phase to each of these tubes. Mark the level of the lower phase on the tube before loading the upper phase.Then carefully stack five milliliters of the upper phase unto the lower phase, pipetting it very slowly down along the walls of the tube. When the gradients are prepared and centrifugation is complete load the supernatant from the sample equally over the two gradients. Fill the remaining space in each gradient tube with Ad-buffer containing ten millimolar TCEP making sure to fill the tubes all the way to the top.Adjust the weight of the opposite tubes to a zero weight difference. And alter centrifuge for 176000 times G and 4 degrees Celsius for two hours. After this, use a clamp to fix one ultra centrifugation tubes to a stand.Making sure to leave the area around the lower phase level marked accessible. Place the goose-necked lamp above the tube. Around the mark, at the border between the lower and upper phases, a distinct band should be visible.Puncture the tube with a needle, and aspirate the vector band into a syringe to collect the vector. Transfer collected vector virons to a 15 milliliter tube and calculate the amount of coupling substrate needed as outlined in the text protocol. Then, transfer the vector solution to a tube of appropriate size.And the calculated amount of coupling compounds stock solution. Gently mix by overhead rotation for one hour at room temperature. Next, adjust the total volume of the vector solution to six milliliters by adding Ad buffer.Purify the vector from the unreacted coupling moiety using a second Cesium chloride binding step as outlined in the text protocol. Puncture the tube with a needle and aspirate the vector band into a syringe. Transfer the collected vectors to a 15 milliliter tube.If the combined from both gradient tubes is less than 2.5 milliliters, adjust the volume to 2.5 milliliters by adding Ad buffer. Load the combined virons unto an equal upgraded Pd column and discard the flow through. Then add three milliliters of Ad buffer to the column and collect the flow through.Add 333 microliters of glycerol to the collected flow through to obtain the final concentration of ten percent. Divide these into suitable aliquots. Making sure to include an aliquot of 20 microliters for tighter determination by OD 260 measurement.Store the vector solution at 80 degrees Celsius until ready to use. Representative results of the effects on 293 cells indicate that vector production is successful. Cells should show morphology 40-48 hours after being inoculated with the virus vector.Silver stained SDS phage is then used to assessed coupling efficiency. Vectors with Cysteins introduced into the capsomere pentone base both with the hexon modified and unmodified are run. The Hexon bands exhibit a shift indicating successful modification of over 90 percent of the monomers per capsid.While attempting this procedure it is important to ensure a reducing environment for unmodofied vector particle. After modification, a regular atmosphere is suitable. Precise documentary calculations have to ensure quantitative modification of all vector particles.The development of this technique helps research in the field of Gene Therapy to further explore safe and deficient strategies for vector delivery. Following this procedure other methods like coupling of ligands for targeting or fluorescent dyes for labeling can be performed in order to answer additional questions like receptor usage and bio distribution. Please make sure to obey your local GMO and Working Safety Regulations.

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8.2K visualizações.  University Witten/Herdecke. Este método pode ajudar a responder perguntas-chave no campo da Terapia Genética, Oncolise e Vacinação Genética. Facilita a compreensão e modulação das interações de vetores dos vetores de transferência de genes adenovírus, o tipo vetorial mais usado em ensaios clínicos atualizados. A principal vantagem desta técnica é que os capsulados de adenovírus podem ser especificamente modificados de forma altamente definida em posições selecionadas e em proporção variável usando q...