Vector Affinity and Receptor Distribution Define Tissue-Specific Targeting in an Engineered AAV Capsid.

Unbiased in vivo selections of diverse capsid libraries can yield engineered capsids that overcome gene therapy delivery challenges like traversing the blood-brain barrier (BBB), but little is known about the parameters of capsid-receptor interactions that govern their improved activity. This hampers broader efforts in precision capsid engineering and is a practical impediment to ensuring the translatability of capsid properties between preclinical animal models and human clinical trials. In this work, we utilize the adeno-associated virus (AAV)-PHP.B-Ly6a model system to better understand the targeted delivery and BBB penetration properties of AAV vectors. This model offers a defined capsid-receptor pair that can be used to systematically define relationships between target receptor affinity and in vivo activity of engineered AAV vectors. Here, we report a high-throughput method for quantifying capsid-receptor affinity and demonstrate that direct binding assays can be used to organize a vector library into families with varied affinity for their target receptor. Our data indicate that efficient central nervous system transduction requires high levels of target receptor expression at the BBB, but it is not a requirement for receptor expression to be limited to the target tissue. We observed that enhanced receptor affinity leads to reduced transduction of off-target tissues but can negatively impact on-target cellular transduction and penetration of endothelial barriers. Together, this work provides a set of tools for defining vector-receptor affinities and demonstrates how receptor expression and affinity interact to impact the performance of engineered AAV vectors in targeting the central nervous system. IMPORTANCE Novel methods for measuring adeno-associated virus (AAV)-receptor affinities, especially in relation to vector performance in vivo, would be useful to capsid engineers as they develop AAV vectors for gene therapy applications and characterize their interactions with native or engineered receptors. Here, we use the AAV-PHP.B-Ly6a model system to assess the impact of receptor affinity on the systemic delivery and endothelial penetration properties of AAV-PHP.B vectors. We discuss how receptor affinity analysis can be used to isolate vectors with optimized properties, improve the interpretation of library selections, and ultimately translate vector activities between preclinical animal models and humans.

Intranasal gene therapy to prevent infection by SARS-CoV-2 variants.

SARS-CoV-2 variants have emerged with enhanced pathogenicity and transmissibility, and escape from pre-existing immunity, suggesting first-generation vaccines and monoclonal antibodies may now be less effective. Here we present an approach for preventing clinical sequelae and the spread of SARS-CoV-2 variants. First, we affinity matured an angiotensin-converting enzyme 2 (ACE2) decoy protein, achieving 1000-fold binding improvements that extend across a wide range of SARS-CoV-2 variants and distantly related, ACE2-dependent coronaviruses. Next, we demonstrated the expression of this decoy in proximal airway when delivered via intranasal administration of an AAV vector. This intervention significantly diminished clinical and pathologic consequences of SARS-CoV-2 challenge in a mouse model and achieved therapeutic levels of decoy expression at the surface of proximal airways when delivered intranasally to nonhuman primates. Importantly, this long-lasting, passive protection approach is applicable in vulnerable populations such as the elderly and immune-compromised that do not respond well to traditional vaccination. This approach could be useful in combating COVID-19 surges caused by SARS-CoV-2 variants and should be considered as a countermeasure to future pandemics caused by one of the many pre-emergent, ACE2-dependent CoVs that are poised for zoonosis.

Context-Specific Function of the Engineered Peptide Domain of PHP.B.

One approach to improve the utility of adeno-associated virus (AAV)-based gene therapy is to engineer the AAV capsid to (i) overcome poor transport through tissue barriers and (ii) redirect the broadly tropic AAV to disease-relevant cell types. Peptide- or protein-domain insertions into AAV surface loops can achieve both engineering goals by introducing a new interaction surface on the AAV capsid. However, we understand little about the impact of insertions on capsid structure and the extent to which engineered inserts depend on a specific capsid context to function. Here, we examine insert-capsid interactions for the engineered variant AAV9-PHP.B. The 7-amino-acid peptide insert in AAV9-PHP.B facilitates transport across the murine blood-brain barrier via binding to the receptor Ly6a. When transferred to AAV1, the engineered peptide does not bind Ly6a. Comparative structural analysis of AAV1-PHP.B and AAV9-PHP.B revealed that the inserted 7-amino-acid loop is highly flexible and has remarkably little impact on the surrounding capsid conformation. Our work demonstrates that Ly6a binding requires interactions with both the PHP.B peptide and specific residues from the AAV9 HVR VIII region. An AAV1-based vector that incorporates a larger region of AAV9-PHP.B-including the 7-amino-acid loop and adjacent HVR VIII amino acids-can bind to Ly6a and localize to brain tissue. However, unlike AAV9-PHP.B, this AAV1-based vector does not penetrate the blood-brain barrier. Here we discuss the implications for AAV capsid engineering and the transfer of engineered activities between serotypes. IMPORTANCE Targeting AAV vectors to specific cellular receptors is a promising strategy for enhancing expression in target cells or tissues while reducing off-target transgene expression. The AAV9-PHP.B/Ly6a interaction provides a model system with a robust biological readout that can be interrogated to better understand the biology of AAV vectors' interactions with target receptors. In this work, we analyzed the sequence and structural features required to successfully transfer the Ly6a receptor-binding epitope from AAV9-PHP.B to another capsid of clinical interest, AAV1. We found that AAV1- and AAV9-based vectors targeted to the same receptor exhibited different brain-transduction profiles. Our work suggests that, in addition to attachment-receptor binding, the capsid context in which this binding occurs is important for a vector's performance.

BID preferentially activates BAK while BIM preferentially activates BAX, affecting chemotherapy response.

Apoptosis is a highly regulated form of cell death that controls normal homeostasis as well as the antitumor activity of many chemotherapeutic agents. Commitment to death via the mitochondrial apoptotic pathway requires activation of the mitochondrial pore-forming proteins BAK or BAX. Activation can be effected by the activator BH3-only proteins BID or BIM, which have been considered to be functionally redundant in this role. Herein, we show that significant activation preferences exist between these proteins: BID preferentially activates BAK while BIM preferentially activates BAX. Furthermore, we find that cells lacking BAK are relatively resistant to agents that require BID activation for maximal induction of apoptosis, including topoisomerase inhibitors and TRAIL. Consequently, patients with tumors that harbor a loss of BAK1 exhibit an inferior response to topoisomerase inhibitor treatment in the clinic. Therefore, BID and BIM have nonoverlapping roles in the induction of apoptosis via BAK and BAX, affecting chemotherapy response.

The GPI-Linked Protein LY6A Drives AAV-PHP.B Transport across the Blood-Brain Barrier.

Efficient delivery of gene therapy vectors across the blood-brain barrier (BBB) is the holy grail of neurological disease therapies. A variant of the neurotropic vector adeno-associated virus (AAV) serotype 9, called AAV-PHP.B, was shown to very efficiently deliver transgenes across the BBB in C57BL/6J mice. Based on our recent observation that this phenotype is mouse strain dependent, we used whole-exome sequencing-based genetics to map this phenotype to a specific haplotype of lymphocyte antigen 6 complex, locus A (Ly6a) (stem cell antigen-1 [Sca-1]), which encodes a glycosylphosphatidylinositol (GPI)-anchored protein whose function had been thought to be limited to the biology of hematopoiesis. Additional biochemical and genetic studies definitively linked high BBB transport to the binding of AAV-PHP.B with LY6A (SCA-1). These studies identify, for the first time, a ligand for this GPI-anchored protein and suggest a role for it in BBB transport that could be hijacked by viruses in natural infections or by gene therapy vectors to treat neurological diseases.

Deamidation of Amino Acids on the Surface of Adeno-Associated Virus Capsids Leads to Charge Heterogeneity and Altered Vector Function.

Post-translational modification of the adeno-associated virus capsids is a poorly understood factor in the development of these viral vectors into pharmaceutical products. Here we report the extensive capsid deamidation of adeno-associated virus serotype 8 and seven other diverse adeno-associated virus serotypes, with supporting evidence from structural, biochemical, and mass spectrometry approaches. The extent of deamidation at each site depended on the vector's age and multiple primary-sequence and three-dimensional structural factors. However, the extent of deamidation was largely independent of the vector recovery and purification conditions. We demonstrate the potential for deamidation to impact transduction activity and, moreover, correlate an early time point loss in vector activity to rapidly progressing spontaneous deamidation at several adeno-associated virus 8 asparagines. We explore mutational strategies that stabilize side-chain amides, improving vector transduction and reducing the lot-to-lot molecular variability that presents a key concern in biologics manufacturing. This study illuminates a previously unknown aspect of adeno-associated virus capsid heterogeneity and highlights its importance in the development of these vectors for gene therapy.

Linkage-specific avidity defines the lysine 63-linked polyubiquitin-binding preference of rap80.

Linkage-specific polyubiquitin recognition is thought to make possible the diverse set of functional outcomes associated with ubiquitination. Thus far, mechanistic insight into this selectivity has been largely limited to single domains that preferentially bind to lysine 48-linked polyubiquitin (K48-polyUb) in isolation. Here, we propose a mechanism, linkage-specific avidity, in which multiple ubiquitin-binding domains are arranged in space so that simultaneous, high-affinity interactions are optimum with one polyUb linkage but unfavorable or impossible with other polyUb topologies and monoUb. Our model is human Rap80, which contains tandem ubiquitin interacting motifs (UIMs) that bind to K63-polyUb at DNA double-strand breaks. We show how the sequence between the Rap80 UIMs positions the domains for efficient avid binding across a single K63 linkage, thus defining selectivity. We also demonstrate K48-specific avidity in a different protein, ataxin-3. Using tandem UIMs, we establish the general principles governing polyUb linkage selectivity and affinity in multivalent ubiquitin receptors.

Fractional killing arises from cell-to-cell variability in overcoming a caspase activity threshold.

When cells are exposed to death ligands such as TRAIL, a fraction undergoes apoptosis and a fraction survives; if surviving cells are re-exposed to TRAIL, fractional killing is once again observed. Therapeutic antibodies directed against TRAIL receptors also cause fractional killing, even at saturating concentrations, limiting their effectiveness. Fractional killing arises from cell-to-cell fluctuations in protein levels (extrinsic noise), but how this results in a clean bifurcation between life and death remains unclear. In this paper, we identify a threshold in the rate and timing of initiator caspase activation that distinguishes cells that live from those that die; by mapping this threshold, we can predict fractional killing of cells exposed to natural and synthetic agonists alone or in combination with sensitizing drugs such as bortezomib. A phenomenological model of the threshold also quantifies the contributions of two resistance genes (c-FLIP and Bcl-2), providing new insight into the control of cell fate by opposing pro-death and pro-survival proteins and suggesting new criteria for evaluating the efficacy of therapeutic TRAIL receptor agonists.

Polyubiquitin-sensor proteins reveal localization and linkage-type dependence of cellular ubiquitin signaling.

Polyubiquitin chain topology is thought to direct modified substrates to specific fates, but this function-topology relationship is poorly understood, as are the dynamics and subcellular locations of specific polyubiquitin signals. Experimental access to these questions has been limited because linkage-specific inhibitors and in vivo sensors have been unavailable. Here we present a general strategy to track linkage-specific polyubiquitin signals in yeast and mammalian cells, and to probe their functions. We designed several high-affinity Lys63 polyubiquitin-binding proteins and demonstrate their specificity in vitro and in cells. We apply these tools as competitive inhibitors to dissect the polyubiquitin-linkage dependence of NF-κB activation in several cell types, inferring the essential role of Lys63 polyubiquitin for signaling via the IL-1ß and TNF-related weak inducer of apoptosis (TWEAK) but not TNF-α receptors. We anticipate live-cell imaging, proteomic and biochemical applications for these tools and extension of the design strategy to other polymeric ubiquitin-like protein modifications.

High activity of an affinity-matured ACE2 decoy against Omicron SARS-CoV-2 and pre-emergent coronaviruses.

The viral genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), particularly its cell-binding spike protein gene, has undergone rapid evolution during the coronavirus disease 2019 (COVID-19) pandemic. Variants including Omicron BA.1 and Omicron BA.2 now seriously threaten the efficacy of therapeutic monoclonal antibodies and vaccines that target the spike protein. Viral evolution over a much longer timescale has generated a wide range of genetically distinct sarbecoviruses in animal populations, including the pandemic viruses SARS-CoV-2 and SARS-CoV-1. The genetic diversity and widespread zoonotic potential of this group complicates current attempts to develop drugs in preparation for the next sarbecovirus pandemic. Receptor-based decoy inhibitors can target a wide range of viral strains with a common receptor and may have intrinsic resistance to escape mutant generation and antigenic drift. We previously generated an affinity-matured decoy inhibitor based on the receptor target of the SARS-CoV-2 spike protein, angiotensin-converting enzyme 2 (ACE2), and deployed it in a recombinant adeno-associated virus vector (rAAV) for intranasal delivery and passive prophylaxis against COVID-19. Here, we demonstrate the exceptional binding and neutralizing potency of this ACE2 decoy against SARS-CoV-2 variants including Omicron BA.1 and Omicron BA.2. Tight decoy binding tracks with human ACE2 binding of viral spike receptor-binding domains across diverse clades of coronaviruses. Furthermore, in a coronavirus that cannot bind human ACE2, a variant that acquired human ACE2 binding was bound by the decoy with nanomolar affinity. Considering these results, we discuss a strategy of decoy-based treatment and passive protection to mitigate the ongoing COVID-19 pandemic and future airway virus threats.

Avid interactions underlie the Lys63-linked polyubiquitin binding specificities observed for UBA domains.

Ubiquitin (denoted Ub) receptor proteins as a group must contain a diverse set of binding specificities to distinguish the many forms of polyubiquitin (polyUb) signals. Previous studies suggested that the large class of ubiquitin-associated (UBA) domains contains members with intrinsic specificity for Lys63-linked polyUb or Lys48-linked polyUb, thus explaining how UBA-containing proteins can mediate diverse signaling events. Here we show that previously observed Lys63-polyUb selectivity in UBA domains is the result of an artifact in which the dimeric fusion partner, glutathione S-transferase (GST), positions two UBAs for higher affinity, avid interactions with Lys63-polyUb, but not with Lys48-polyUb. Freed from GST, these UBAs are either nonselective or prefer Lys48-polyUb. Accordingly, NMR experiments reveal no Lys63-polyUb-specific binding epitopes for these UBAs. We reexamine previous conclusions based on GST-UBAs and present an alternative model for how UBAs achieve a diverse range of linkage specificities.