Aravind Asokan

Adeno-associated viruses (AAV) are non-enveloped parvoviruses widely utilized for therapeutic gene delivery. The membrane-associated accessory protein (MAAP), a key viral protein, is critical for cellular egress of both infectious and recombinant AAV particles. However, the structure-function correlates of this process have remained largely elusive. Here, we applied structure-guided evolution to enhance AAV egress and dissect the biology of both natural and synthetic MAAP (synMAAP) variants. Three structurally distinct combinatorial libraries focused on disordered domains within MAAP8 (derived from AAV serotype 8) were subjected to selection through both transfection and infectious cycling. Newly enriched synMAAPs obtained as a function of enhanced AAV secretion from host cells revealed a profound bias toward mutations within the C-terminal domain. The most potent synMAAPs showed increased propensity to form extended alpha-helix structures as predicted by structural modeling, amphipathicity, and hydrophobicity scores. Confocal microscopy demonstrated that, unlike natural MAAP8, synMAAPs are excluded from the nucleus and exhibit strong perinuclear accumulation within the Golgi. Proximity ligation analysis, biochemical analysis, and gene deletion studies further corroborated that synMAAPs appear to more efficiently engage SNARE complex proteins, notably involving VAMP3, to potentiate AAV egress. Together, these findings reveal new insights into structure-function correlates of viral egress from host cells and may help improve recombinant vector production for gene therapy applications. IMPORTANCE: Viruses must exit infected cells to spread, yet this process is poorly understood for adeno-associated virus. In this study, we examine how adeno-associated virus exits mammalian cells without causing cell death. By engineering and evolving new versions of the viral egress factor, we identify cellular trafficking pathways that are exploited during viral release. These findings improve our understanding of how viruses interact with host cells. Additionally, the engineered egress factors described in this study may be used to improve the efficiency of adeno-associated virus production for gene therapy applications.

Adeno-associated virus (AAV) vectors are promising vehicles for the delivery and long-term expression of antibodies for treatment of chronic diseases. Given the strong link between the C-C chemokine receptor 5 (CCR5)–Δ 32/ Δ 32 genotype and protection from HIV, we explored the ability of AAV vectors expressing the CCR5-blocking antibody leronlimab to mediate a functional cure in simian-human immunodeficiency virus (SHIV)–infected rhesus macaques by interrupting viral access to the viral entry co-receptor CCR5. Delivery of AAV-leronlimab to rhesus macaques elicited antidrug antibodies (ADA) and rapid plasma leronlimab clearance in approximately half of the treated macaques. However, in these same animals, we observed spontaneous leronlimab transgene reemergence and detectable CCR5 receptor occupancy approximately 1 year later that subsequently persisted indefinitely without rebound of ADA. In macaques that did not mount robust ADA responses, detectable plasma leronlimab concentrations and CCR5 receptor occupancy were maintained for more than 1 year. Of the nine macaques producing sufficient leronlimab to achieve full CCR5 receptor occupancy on blood CD4 ⁺ T cells, AAV-leronlimab drove stringent or partial control of SHIV viremia in six macaques long term. In the three macaques with uncontrolled SHIV viremia, short-term administration of exogenous leronlimab induced complete SHIV suppression in two and a 100-fold reduction in the third, indicating that a threshold of leronlimab expression is necessary to effectively halt SHIV replication. These results demonstrate the potential of gene therapy–mediated long-term antibody-based CCR5 blockade for HIV functional cure but highlight challenges in achieving sufficient antibody expression when targeting an abundant self-antigen.

Limb loss remains a significant clinical challenge, but regenerative medicine approaches such as gene therapy offer a promising strategy to trigger endogenous regeneration programs. Optimal vector configurations and molecular targets for appendicular skeletal repair are not well defined. Here, we leveraged insights from species with a high endogenous capacity for appendage regeneration to design an enhancer-directed gene delivery platform that functions during mouse digit regeneration, a well-characterized model for partial limb regeneration in mammals. Single-cell RNA sequencing of zebrafish caudal fin regeneration, combined with expression data in regenerating salamander limbs and mouse digit tips, implicated the SP family of transcription factors as conserved, epidermally expressed mediators of appendage regrowth. Null mutants of Sp8 demonstrated impaired limb regeneration in salamanders, while conditional knockout of Sp6 and/or Sp8 in the mouse basal epidermis resulted in defective bony digit tip regeneration, involving an IL-17-mediated osteoclastogenic program. Spatiotemporally focused expression of FGF8, a known target of SP factors, using a zebrafish-derived tissue regeneration enhancer element via adeno-associated viral vectors, could partially rescue digit tip regeneration in SP knockout mice and accelerate digit regeneration in wild-type mice. Our results demonstrate a contextual gene therapy approach to address limb loss based on genes like SP transcription factors conserved across multiple contexts of appendage regeneration.

Engineered T cells, reprogrammed to express chimeric antigen receptors (CAR) or T cell receptors (TCR), have transformed cancer treatment and are being explored as therapeutics for autoimmune and infectious diseases. Enhancing T cell function through genome editing, either by disrupting endogenous genes or precisely inserting DNA payloads, has shown considerable promise ¹ . However, the ex vivo manufacturing process is lengthy and costly, limiting accessibility of these therapies. In vivo generation of CAR T cells could overcome these barriers, but current methods rely either on transient expression with limited durability, or on random integration of DNA payloads that lack specificity. Here we demonstrate that stable and cell-specific transgene expression can be achieved through in vivo site-specific integration of large DNA payloads. We developed a two-vector system to deliver CRISPR–Cas9 ribonucleoproteins and a DNA donor template, using enveloped delivery vehicles and adeno-associated viruses, respectively. We optimized both vectors for T cell-specific delivery and gene-targeting efficiency. By integrating a CAR transgene into a T cell-specific locus, we generate therapeutic levels of CAR T cells in vivo in humanized mouse models of B cell aplasia, and haematological and solid malignancies. These findings offer a pathway to more efficient, precise and widely accessible T cell therapies.

Glycogen storage disease (GSD) type IX γ2 is a rare inborn error of metabolism where a defect in glycogenolysis leads to the inability to break down glycogen in the liver. Patients with GSD IX γ2 develop hypoglycemia and advanced liver disease, placing them at risk for liver transplantation. This study evaluates the efficacy of liver-directed AAV gene therapy in a murine model of GSD IX γ2. Phkg2 ^−/− mice underwent treatment with AAV gene therapy (AAV9-LSP- mPhkg2 , 5 × 10 ¹² vg/kg, intravenous delivery) at ages 3 or 6 months and were treated for either 2 weeks, 3 months, or 12 months. Results demonstrated that AAV gene therapy reduced GSD IX γ2 disease burden across all primary end points. AAV gene therapy also persisted across the mouse lifespan and reduced preexisting liver fibrosis. This work provides preclinical data supporting AAV gene therapy as a definitive treatment for GSD IX γ2.