Post‐translational modifications in viral capsids are known to fine‐tune and regulate several aspects of the infective life cycle of several viruses in the host. Recombinant viruses that are generated in a specific producer cell line are likely to inherit unique post‐translational modifications during intra‐cellular maturation of its capsid proteins. Data on such post‐translational modifications in the capsid of recombinant adeno‐associated virus serotypes (
AAV
1‐rh10) is limited. We have employed liquid chromatography and mass spectrometry analysis to characterize post‐translational modifications in
AAV
1‐rh10 capsid protein. Our analysis revealed a total of 52 post‐translational modifications in
AAV
2‐
AAV
rh10 capsids, including ubiquitination (17%), glycosylation (36%), phosphorylation (21%),
SUMO
ylation (13%) and acetylation (11%). While
AAV
1 had no detectable post‐translational modification, at least four
AAV
serotypes had >7 post‐translational modifications in their capsid protein. About 82% of these post‐translational modifications are novel. A limited validation of
AAV
2 capsids by
MALDI
‐
TOF
and western blot analysis demonstrated minimal glycosylation and ubiquitination of
AAV
2 capsids. To further validate this, we disrupted a glycosylation site identified in
AAV
2 capsid (
AAV
2‐N253Q), which severely compromised its packaging efficiency (~ 100‐fold vs.
AAV
2 wild‐type vectors). In order to confirm other post‐translational modifications detected such as
SUMO
ylation, mutagenesis of a
SUMO
ylation site(K258Q) in
AAV
2 was performed. This mutant vector demonstrated reduced levels of
SUMO
‐1/2/3 proteins and negligible transduction, 2 weeks after ocular gene transfer. Our study underscores the heterogeneity of post‐translational modifications in
AAV
vectors. The data presented here, should facilitate further studies to understand the biological relevance of post‐translational modifications in
AAV
life cycle and the development of novel bioengineered
AAV
vectors for gene therapy applications.
Enzymes
Trypsin,
EC 3.4.21.4
In the majority of patients ASCAs disappeared during a GFD. In children this disappearance of ASCA positivity was more pronounced. This can be explained by the well-known fact that gut permeability normalizes much better in children than in adults. Also, the adults had higher levels of ASCAs at diagnosis. This was probably because they had been exposed to gluten for longer and therefore had more long-lasting damage.
Recombinant
adeno-associated virus (AAV)-based gene therapy has
been promising, but several host-related transduction or immune challenges
remain. For this mode of therapy to be widely applicable, it is crucial
to develop high transduction and permeating vectors that infect the
target at significantly low doses. Because glycosylation of capsid
proteins is known to be rate limiting in the life cycle of many viruses,
we reasoned that perturbation of glycosylation sites in AAV2 capsid
will enhance gene delivery. In our first set experiments, pharmacological
modulation of the glycosylation status in host cells, modestly decreased
(1-fold) AAV2 packaging efficacy while it improved their gene expression
(∼74%) in vitro. We then generated 24 mutant AAV2 vectors modified
to potentially create or disrupt a glycosylation site in its capsid.
Three of them demonstrated a 1.3–2.5-fold increase in transgene
expression in multiple cell lines (HeLa, Huh7, and ARPE-19). Hepatic
gene transfer of these vectors in hemophilia B mice, resulted in a
2-fold increase in human coagulation factor (F)IX levels, while its
T/B-cell immunogenic response was unaltered. Subsequently, intravitreal
gene transfer of glycosylation site-modified vectors in C57BL6/J mice
demonstrated an increase in green fluorescence protein expression
(∼2- to 4-fold) and enhanced permeation across retina. Subretinal
administration of these modified vectors containing RPE65 gene further
rescued the photoreceptor response in a murine model of Leber congenital
amarousis. Our studies highlight the translational potential of glycosylation
site-modified AAV2 vectors for hepatic and ocular gene therapy applications.
Synthetic engineering of viral vectors such as adeno-associated virus (AAV) is crucial to overcome host transduction barriers observed during clinical gene therapy. We reasoned that exploring the role of cellular ubiquitin-like modifiers (UBLs) such as Neddylation or SUMOylation during AAV transduction could be beneficial. Using a combination of in silico biochemical and molecular engineering strategies, we have studied the impact of these UBLs during AAV2 infection and further developed Neddylation or SUMOylation site–modified AAV vectors and validated them in multiple disease models in vitro and in vivo. Hepatic gene transfer of two novel vectors developed, K105Q (SUMOylation-site mutant) and K665Q (Neddylation-site mutant), demonstrated a significantly improved human coagulation factor (F) IX expression (up to two-fold) in a murine model of hemophilia B. Furthermore, subretinal gene transfer of AAV2-K105Q vector expressing RPE65 gene demonstrated visual correction in a murine model of a retinal degenerative disease (rd12 mice). These vectors did not have any adverse immunogenic events in vivo. Taken together, we demonstrate that gene delivery vectors specifically engineered at UBLs can improve the therapeutic outcome during AAV-mediated ocular or hepatic gene therapy.
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