Site-specific modifications to AAV8 capsid yields enhanced brain transduction in the neonatal MPS IIIB mouse

Abstract

Mucopolysaccharidosis type IIIB (MPS IIIB) is an autosomal recessive lysosomal disease caused by defective production of the enzyme α-N-acetylglucosaminidase. It is characterized by severe and complex central nervous system degeneration. Effective therapies will likely target early onset disease and overcome the blood-brain barrier. Modifications of adenoassociated viral (AAV) vector capsids that enhance transduction efficiency have been described in the retina. Herein, we describe for the first time, a transduction assessment of two intracranially administered adeno-associated virus serotype 8 variants, in which specific surface-exposed tyrosine (Y) and threonine (T) residues were substituted with phenylalanine (F) and valine (V) residues, respectively. A double-mutant (Y444 + 733F) and a triple-mutant (Y444 + 733F + T494V) AAV8 were evaluated for their efficacy for the potential treatment of MPS IIIB in a neonatal setting. We evaluated biodistribution and transduction profiles of both variants compared to the unmodified parental AAV8, and assessed whether the method of vector administration would modulate their utility. Vectors were administered through four intracranial routes: six sites (IC6), thalamic (T), intracerebroventricular, and ventral tegmental area into neonatal mice. Overall, we conclude that the IC6 method resulted in the widest biodistribution within the brain. Noteworthy, we demonstrate that GFP intensity was significantly more robust with AAV8 (double Y-F + TV) compared to AAV8 (double Y-F). This provides proof of concept for the enhanced utility of IC6 administration of the capsid modified AAV8 (double Y-F + TV) as a valid therapeutic approach for the treatment of MPS IIIB, with further implications for other monogenic diseases.

Analysis of Host Cell Proteins in AAV Products with ProteoMiner Protein Enrichment Technology

“…MS-based technologies have been very well developed and widely used in HCP analysis in mAb drugs, but their application of MS on HCP analysis in AAV products is still in its early stage. Most of the HCPs in the gene therapy products were identified by direct digestion followed by LC-MS/MS analysis. − SDS-PAGE and 2D-PAGE coupled with in-gel digestion and LC-MS/MS analysis have been utilized to identify host cell proteins (HCPs) that copurify with adeno-associated virus (AAV) during different purification processes . Recently, a single-pot, solid-phase-enhanced sample-preparation (SP3) technology has been applied to HCP analysis in AAV products .…”

“…Additionally, we found transcriptional proteins, heat shock proteins, immunoglobulins, glycoproteins, and ribonucleoproteins among the HCPs (Table S2). Of the 847 HCPs identified, 263 have been reported in the literature on AAV products produced by the human cell line HEK293. − These proteins include frequently reported HCPs such as nucleophosmin, protein SET, and nucleolin, as well as RNA-binding proteins such as small nuclear ribonucleoproteins Sm D2, D3, heterogeneous nuclear ribonucleoprotein A/B, and PABP. Additionally, some HCPs commonly found in mAbs, such as peroxiredoxin 1, ubiquitin, actin, and Hsc70, which may regulate cell functions, were also detected in AAV products (Figure S4).…”

“…In contrast to the extensive HCP analysis methods used for mAb products, approaches for analyzing HCPs in gene therapy products using MS are very limited. , Most HCP identification in gene therapy products is performed through direct digestion followed by LC-MS/MS analysis. − SDS-PAGE or 2D-PAGE coupled with in-gel digestion and LC-MS/MS has also been applied to separate HCPs from capsid proteins, thereby enabling detection of low-abundance HCPs. , To date, only one enrichment method has been reported to decrease the dynamic range between AAV capsid protein peptides and HCP peptides using SP3-based technology . A major challenge in HCP analysis in gene therapy is limited product quality.…”

AAV Engineering for Improving Tropism to the Central Nervous System

“…These efforts were initially serotype specific, leading to enhanced AAV2 neuronal transduction in hippocampal and striatal regions [13,14,21]. After an improved understanding of ancestral cap sequences, the disruption of native cellular binding motifs led to improved CNS transduction with AAV6 [22], AAV8 [23][24][25][26], and AAV9 serotypes [27,28]. Similar mutagenesis techniques were employed on surface residues to evade the ubiquitin-proteasome pathway, a potent obstacle to proper AAV intracellular trafficking [28].…”

“…These engineered capsids led to a significant reduction in virion ubiquitination, mitigating target recognition for proteasomal degradation, and prolonging efficacy durability [21]. This approach accelerated the infective capabilities of AAV vectors by increasing efficient second-strand synthesis and subsequent nuclear transport [15,17,22,25].…”