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Tailoring the AAV2 capsid vector for bone-targeting.

ABSTRACT: BACKGROUND:Targeting specific tissues remains a major challenge to the promise of gene therapy. For example, several strategies have failed to target adeno-associated virus 2 (AAV2) vectors, to bone. We have evaluated in vitro and in vivo the affinity of an AAV2 vector to bone matrix, hydroxyapatite (HA) to treat Mucopolysacccharidosis IVA. METHODS:To increase vector affinity to HA, an aspartic acid octapeptide (D8) was inserted immediately after the N-terminal region of the VP2 capsid protein. The modified vector had physical titers and transduction efficiencies comparable to the unmodified vector. RESULTS:The bone-targeting vector had significantly higher HA affinity and vector genome copies in bone than the unmodified vector. The modified vector was also released from HA, and its enzyme activity in bone, 3 months post infusion, was 4.7-fold higher than the unmodified vector. CONCLUSION:Inserting a bone-targeting peptide into the vector capsid increases gene delivery and expression in the bone without decreasing enzyme expression. This approach could be a novel strategy to treat systemic bone diseases.

SUBMITTER: Almeciga-Diaz CJ

PROVIDER: S-EPMC6266866 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Tailoring the AAV2 capsid vector for bone-targeting.

Alméciga-Díaz Carlos J CJ Montaño Adriana M AM Barrera Luis A LA Tomatsu Shunji S

Pediatric research 20181015 4

<h4>Background</h4>Targeting specific tissues remains a major challenge to the promise of gene therapy. For example, several strategies have failed to target adeno-associated virus 2 (AAV2) vectors, to bone. We have evaluated in vitro and in vivo the affinity of an AAV2 vector to bone matrix, hydroxyapatite (HA) to treat Mucopolysacccharidosis IVA.<h4>Methods</h4>To increase vector affinity to HA, an aspartic acid octapeptide (D8) was inserted immediately after the N-terminal region of the VP2 c ...[more]

PMID: 30323349

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Project description:Adeno-associated virus type 2 (AAV2) has proven to be a valuable vector for gene therapy. Characterization of the functional domains of the AAV capsid proteins can facilitate our understanding of viral tissue tropism, immunoreactivity, viral entry, and DNA packaging, all of which are important issues for generating improved vectors. To obtain a comprehensive genetic map of the AAV capsid gene, we have constructed 93 mutants at 59 different positions in the AAV capsid gene by site-directed mutagenesis. Several types of mutants were studied, including epitope tag or ligand insertion mutants, alanine scanning mutants, and epitope substitution mutants. Analysis of these mutants revealed eight separate phenotypes. Infectious titers of the mutants revealed four classes. Class 1 mutants were viable, class 2 mutants were partially defective, class 3 mutants were temperature sensitive, and class 4 mutants were noninfectious. Further analysis revealed some of the defects in the class 2, 3, and 4 mutants. Among the class 4 mutants, a subset completely abolished capsid formation. These mutants were located predominantly, but not exclusively, in what are likely to be beta-barrel structures in the capsid protein VP3. Two of these mutants were insertions at the N and C termini of VP3, suggesting that both ends of VP3 play a role that is important for capsid assembly or stability. Several class 2 and 3 mutants produced capsids that were unstable during purification of viral particles. One mutant, R432A, made only empty capsids, presumably due to a defect in packaging viral DNA. Additionally, five mutants were defective in heparan binding, a step that is believed to be essential for viral entry. These were distributed into two amino acid clusters in what is likely to be a cell surface loop in the capsid protein VP3. The first cluster spanned amino acids 509 to 522; the second was between amino acids 561 and 591. In addition to the heparan binding clusters, hemagglutinin epitope tag insertions identified several other regions that were on the surface of the capsid. These included insertions at amino acids 1, 34, 138, 266, 447, 591, and 664. Positions 1 and 138 were the N termini of VP1 and VP2, respectively; position 34 was exclusively in VP1; the remaining surface positions were located in putative loop regions of VP3. The remaining mutants, most of them partially defective, were presumably defective in steps of viral entry that were not tested in the preliminary screening, including intracellular trafficking, viral uncoating, or coreceptor binding. Finally, in vitro experiments showed that insertion of the serpin receptor ligand in the N-terminal regions of VP1 or VP2 can change the tropism of AAV. Our results provide information on AAV capsid functional domains and are useful for future design of AAV vectors for targeting of specific tissues.

Project description:Adeno-associated virus (AAV) requires co-infection with helper virus for efficient replication. We previously reported that Human Bocavirus 1 (HBoV1) genes, including NP1, NS2, and BocaSR, were critical for AAV2 replication. Here, we first demonstrate the essential roles of the NP1 protein in AAV2 DNA replication and protein expression. We show that NP1 binds to single-strand DNA (ssDNA) at least 30 nucleotides (nt) in length in a sequence-independent manner. Furthermore, NP1 colocalized with the BrdU-labeled AAV2 DNA replication center, and the loss of the ssDNA-binding ability of NP1 by site-directed mutation completely abolished AAV2 DNA replication. We used affinity-tagged NP1 protein to identify host cellular proteins associated with NP1 in cells cotransfected with the HBoV1 helper genes and AAV2 duplex genome. Of the identified proteins, we demonstrate that NP1 directly binds to the DBD-F domain of the RPA70 subunit with a high affinity through the residues 101-121. By reconstituting the heterotrimer protein RPA in vitro using gel filtration, we demonstrate that NP1 physically associates with RPA to form a heterologous complex characterized by typical fast-on/fast-off kinetics. Following a dominant-negative strategy, we found that NP1-RPA complex mainly plays a role in expressing AAV2 capsid protein by enhancing the transcriptional activity of the p40 promoter. Our study revealed a novel mechanism by which HBoV1 NP1 protein supports AAV2 DNA replication and capsid protein expression through its ssDNA-binding ability and direct interaction with RPA, respectively.IMPORTANCERecombinant adeno-associated virus (rAAV) vectors have been extensively used in clinical gene therapy strategies. However, a limitation of these gene therapy strategies is the efficient production of the required vectors, as AAV alone is replication-deficient in the host cells. HBoV1 provides the simplest AAV2 helper genes consisting of NP1, NS2, and BocaSR. An important question regarding the helper function of HBoV1 is whether it provides any direct function that supports AAV2 DNA replication and protein expression. Also of interest is how HBoV1 interplays with potential host factors to constitute a permissive environment for AAV2 replication. Our studies revealed that the multifunctional protein NP1 plays important roles in AAV2 DNA replication via its sequence-independent ssDNA-binding ability and in regulating AAV2 capsid protein expression by physically interacting with host protein RPA. Our findings present theoretical guidance for the future application of the HBoV1 helper genes in the rAAV vector production.

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Tailoring the AAV2 capsid vector for bone-targeting.

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Tailoring the AAV2 capsid vector for bone-targeting.

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