Mapping the structural determinants responsible for enhanced T cell activation to the immunogenic adeno-associated virus capsid from isolate rhesus 32.33.

Avoiding activation of immunity to vector-encoded proteins is critical to the safe and effective use of adeno-associated viral (AAV) vectors for gene therapy. While commonly used serotypes, such as AAV serotypes 1, 2, 7, 8, and 9, are often associated with minimal and/or dysfunctional CD8(+) T cell responses in mice, the threshold for immune activation appears to be lower in higher-order species. We have modeled this discrepancy within the mouse by identifying two capsid variants with differential immune activation profiles: AAV serotype 8 (AAV8) and a hybrid between natural rhesus isolates AAVrh32 and AAVrh33 (AAVrh32.33). Here, we aimed to characterize the structural determinants of the AAVrh32.33 capsid that augment cellular immunity to vector-encoded proteins or those of AAV8 that may induce tolerance. We hypothesized that the structural domain responsible for differential immune activation could be mapped to surface-exposed regions of the capsid, such as hypervariable regions (HVRs) I to IX of VP3. To test this, a series of hybrid AAV capsids was constructed by swapping domains between AAV8 and AAVrh32.33. By comparing their ability to generate transgene-specific T cells in vivo versus the stability of transgene expression in the muscle, we confirmed that the functional domain lies within the VP3 portion of the capsid. Our studies were able to exclude the regions of VP3 which are not sufficient for augmenting the cellular immune response, notably, HVRs I, II, and V. We have also identified HVR IV as a region of interest in conferring the efficiency and stability of muscle transduction to AAVrh32.33.

Retinal degeneration in Aipl1-deficient mice: a new genetic model of Leber congenital amaurosis.

Leber congenital amaurosis (LCA) is the most severe inherited retinopathy, with the earliest age of onset. Because this currently incurable disease is present from birth and is a relatively rare disorder, the development of animal models that closely resemble the phenotype in patients is especially important. Our previous genetic analyses of LCA patients identified mutations in the aryl-hydrocarbon interacting protein-like 1 (AIPL1) gene. Here we present development of an animal model of AIPL1-associated LCA, the Aipl1-deficient mouse. Aipl1 is expressed at low levels throughout human and mouse retinal development and is rapidly upregulated as photoreceptors differentiate. The mouse displays rapid retinal degeneration and massive Müler cell gliosis, resembling the phenotype of the rd mouse, which is caused by a mutation in the gene for the beta-subunit of the rod-specific phosphodiesterase. We confirm that this phenotype is consistent with the human disease using electroretinograms, and document the disease pathogenesis by analyzing the development of all retinal cell types and synaptogenesis during retinal histogenesis. Ectopic expression of AIPL1 led to deregulated retinal progenitor cell proliferation and alterations in cell fate specification; however, no gross abnormalities of proliferation during retinal development were detected. Data from analysis of proliferation and cell fate specification during retinal development of Aipl1-deficient mice suggests that there may be redundancy or compensation for Aipl1 loss by other related proteins. Because this mouse model closely mimics the human retinopathy caused by homozygous mutations in this gene, it provides a preclinical model for testing therapies to rescue the vision of children whose blindness is caused by AIPL1 mutations.

AAV8 capsid variable regions at the two-fold symmetry axis contribute to high liver transduction by mediating nuclear entry and capsid uncoating.

Adeno-associated virus serotype 8 (AAV8) is a promising vector for liver-directed gene therapy. Although efficient uncoating of viral capsids has been implicated in AAV8׳s robust liver transduction, much about the biology of AAV8 hepatotropism remains unclear. Our study investigated the structural basis of AAV8 liver transduction efficiency by constructing chimeric vector capsids containing sequences derived from AAV8 and AAV2 - a highly homologous yet poorly hepatotropic serotype. Engineered vectors containing capsid variable regions (VR) VII & IX from AAV8 in an AAV2 backbone mediated near AAV8-like transduction in mouse liver, with higher numbers of chimeric genomes detected in whole liver cells and isolated nuclei. Interestingly, chimeric capsids within liver nuclei also uncoated similarly to AAV8 by 6 weeks after administration, in contrast with AAV2, of which a significantly smaller proportion were uncoated. This study links specific AAV capsid regions to the transduction ability of a clinically relevant AAV serotype.

Stem cells, microenvironment mechanics, and growth factor activation.

Physicochemical features of a cell's microenvironment can exert important effects on cell behavior and include the effects of matrix elasticity on cell differentiation processes, but molecular mechanisms are largely mysterious. Here we highlight recent reports of a mechanical dependence to growth factor activation, with a particular focus on release of TGFbeta (Transforming Growth Factor beta) from its large latent complex via forced unfolding. We discuss these processes and pathways in the contexts of matrix adhesion and fluid shearing as they might relate to stem cell differentiation and other mechanisms in development, disease, and repair.