Stabilization of Recombinant Adenovirus: Site-Directed Mutagenesis of Key Asparagine Residues in the Hexon Protein

Blanche, Francis; Cameron, Béatrice; Somarriba, S; Maton, L; Barbot, Anne; Guillemin, Thierry

doi:10.1006/abio.2001.5210

Cited by 12 publications

(15 citation statements)

References 15 publications

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“…Ideally, this will allow the production of programmable viral particles, where chemoselective handles can be placed site-specifically by design. Precedence for this approach can be found with the removal of surface-exposed asparagines, which hydrolyze upon storage, changing the surface charge and physiological behavior of adenoviral vectors (5). A general lack of conservation for these exposed methionine codons indicates that this strategy will likely be successful.…”

Section: Discussionmentioning

confidence: 99%

Unnatural Amino Acid Incorporation onto Adenoviral (Ad) Coat Proteins Facilitates Chemoselective Modification and Retargeting of Ad Type 5 Vectors

Banerjee

Ostapchuk

Hearing

et al. 2011

J Virol

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Surface modification of adenovirus vectors can improve tissue-selective targeting, attenuate immunogenicity, and enable imaging of particle biodistribution, thus significantly improving therapeutic potential. Currently, surface engineering is constrained by a combination of factors, including impact on viral fitness, limited access to functionality, or incomplete control over the site of modification. Here, we report a two-step labeling process involving an initial metabolic placement of a uniquely reactive unnatural amino acid, azidohomoalanine (Aha), followed by highly specific chemical modification. As genetic modification of adenovirus is unnecessary, vector production is exceedingly straightforward. Aha incorporation demonstrated no discernible impact on either virus production or infectivity of the resultant particles. "Click" chemical modification of surface-exposed azides was highly selective, allowing for the attachment of a wide range of functionality. Decoration of human adenovirus type 5 (hAd5) with folate, a known cancer-targeting moiety, provided an ϳ20-fold increase in infection of murine breast cancer cells (4T1) in a folate receptor-dependent manner. This study demonstrates that incorporation of unnatural amino acids can provide a flexible, straightforward route for the selective chemical modification of adenoviral vectors.Adenoviruses (Ad) are widely employed vehicles for gene replacement, cancer gene delivery, and vaccine development. As a result of the broad distribution of the coxsackie-adenovirus receptor (CAR) and significant interactions with alternative receptors, most gene delivery applications benefit from more restrictive vector targeting. As a result, significant effort is being invested in both transductional and transcriptional targeting of these vectors (1, 2, 13). Transductional targeting efforts take advantage of three different methods for capsid remodeling: genetic, noncovalent, and chemical modification. Genetic modification of the coat proteins is the most prevalent method and has been explored at numerous positions on the adenoviral surface; however, due to impact on viral stability and infectivity, most studies focus on the HI loop or C terminus of the fiber protein, the hypervariable region (HVR) within the hexon, and the carboxyl terminus of protein IX (pIX) (4,10,26,35,37). Despite being relatively permissive, genetic alteration of these sites often challenges viral fitness, as evidenced by losses in particle production and infectivity, with such issues determined by the nature and size of modification (12,15,19,22).Noncovalent adenoviral decoration was first demonstrated with chemically modified anti-CAR antibodies. Advantageously, this strategy allows for both detargeting from CARmediated infection and retargeting in a single step (7). Such "molecular bridges" between the vector and the cell-specific receptor consist of two domains; often, the first binds specifically to the primary targeting motif in the knob region, effectively masking it, while the second contains ...

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Section: Discussionmentioning

confidence: 99%

Unnatural Amino Acid Incorporation onto Adenoviral (Ad) Coat Proteins Facilitates Chemoselective Modification and Retargeting of Ad Type 5 Vectors

Banerjee

Ostapchuk

Hearing

et al. 2011

J Virol

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“…Consequently coexisting species with differing morphologies may be distinguished and quantitated with improved accuracy compared to light scattering methods. Combined with additional data, comparisons may be drawn between the degree of aggregation and distribution of rAd species in preparations that contain stabilizers or rAds that contain specific point mutations in structural proteins when investigating approaches to improve the stability (Croyle et al, 1998;Blanche et al, 2001). Although the EM method we have described requires general expertise in electron microscopy, the method and the software are easily learned and can be run by most qualified laboratories.…”

Section: Figmentioning

confidence: 96%

The Use of Field Emission Scanning Electron Microscopy to Assess Recombinant Adenovirus Stability

Obenauer-Kutner¹,

Ihnát²,

Yang³

et al. 2002

Human Gene Therapy

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A field emission scanning electron microscopy (FESEM) method was developed to assess the stability of a recombinant adenovirus (rAd). This method was designed to simultaneously sort, count, and size the total number of rAd viral species observed within an image field. To test the method, a preparation of p53 transgene-expressing recombinant adenovirus (rAd/p53) was incubated at 37 degrees C and the viral particles were evaluated by number, structure, and degree of aggregation as a function of time. Transmission electron microscopy (TEM) was also used to obtain ultrastructural detail. In addition, the infectious activity of the incubated rAd/p53 samples was determined using flow cytometry. FESEM image-analysis revealed that incubation at 37 degrees C resulted in a time-dependent decrease in the total number of detectable single rAd/p53 virus particles and an increase in apparent aggregates composed of more than three adenovirus particles. There was also an observed decrease in both the diameter and perimeter of the single rAd/p53 viral particles. TEM further revealed the accumulation of damaged single particles with time at 37 degrees C. The results of this study demonstrate that FESEM, coupled with sophisticated image analysis, may be an important tool in quantifying the distribution of aggregated species and assessing the overall stability of rAd samples.

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“…They showed that the shift corresponded to an increase in isoaspartate residue formation and thus resolved the long-standing puzzle of why chromatograms exhibit a trailing shoulder in the hexon peak (61). To reduce the rate of Asn deamidation, Blanche et al (8) changed all four accessible Asn residues engaged in Asn-Gly pairs in the Ad5 hexon to Leu (Asn 243, Asn 254, and Asn 436) or Ala (Asn 275). These modifications reduced the microheterogeneity in the recombinant adenovirus without adversely affecting virus infectivity.…”

mentioning

confidence: 99%

“…While monitoring the quality and stability of recombinant adenovirus preparations by anion exchange high performance liquid chromatography, Blanche et al (8) noted that the retention time of the hexon peak increased with storage time. They showed that the shift corresponded to an increase in isoaspartate residue formation and thus resolved the long-standing puzzle of why chromatograms exhibit a trailing shoulder in the hexon peak (61).…”

mentioning

confidence: 99%

Structural and Phylogenetic Analysis of Adenovirus Hexons by Use of High-Resolution X-Ray Crystallographic, Molecular Modeling, and Sequence-Based Methods

Rux

Kuser

Burnett

2003

J Virol

208

193

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A major impediment to the use of adenovirus as a gene therapy vector and for vaccine applications is the host immune response to adenovirus hexon-the major protein component of the icosahedral capsid. A solution may lie in novel vectors with modified or chimeric hexons designed to evade the immune response. To facilitate this approach, we have distinguished the portion of hexon that all serotypes have in common from the hypervariable regions that are responsible for capsid diversity and type-specific immunogenicity. The common hexon core-conserved because it forms the viral capsid-sets boundaries to the regions where modifications can be made to produce nonnative hexons. The core has been defined from the large and diverse set of known hexon sequences by an accurate alignment based on the newly refined crystal structures of human adenovirus types 2 (Ad2) and Ad5 hexon. Comparison of the two hexon models, which are the most accurate so far, reveals that over 90% of the residues in each have three-dimensional positions that closely match. Structures for more distant hexons were predicted by building molecular models of human Ad4, chimpanzee adenovirus (AdC68), and fowl adenovirus 1 (FAV1 or CELO). The five structures were then used to guide the alignment of the 40 full-length (>900 residues) hexon sequences in public databases. Distance-and parsimony-based phylogenetic trees are consistent and reveal evolutionary relationships between adenovirus types that parallel those of their animal hosts. The combination of crystallography, molecular modeling, and phylogenetic analysis defines a conserved molecular core that can serve as the armature for the directed design of novel hexons.

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Stabilization of Recombinant Adenovirus: Site-Directed Mutagenesis of Key Asparagine Residues in the Hexon Protein

Cited by 12 publications

References 15 publications

Unnatural Amino Acid Incorporation onto Adenoviral (Ad) Coat Proteins Facilitates Chemoselective Modification and Retargeting of Ad Type 5 Vectors

Unnatural Amino Acid Incorporation onto Adenoviral (Ad) Coat Proteins Facilitates Chemoselective Modification and Retargeting of Ad Type 5 Vectors

The Use of Field Emission Scanning Electron Microscopy to Assess Recombinant Adenovirus Stability

Structural and Phylogenetic Analysis of Adenovirus Hexons by Use of High-Resolution X-Ray Crystallographic, Molecular Modeling, and Sequence-Based Methods

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