Multimodal imaging of capsid and cargo reveals differential brain targeting and liver detargeting of systemically-administered AAVs.

The development of gene delivery vehicles with high organ specificity when administered systemically is a critical goal for gene therapy. We combine optical and positron emission tomography (PET) imaging of 1) reporter genes and 2) capsid tags to assess the temporal and spatial distribution and transduction of adeno-associated viruses (AAVs). AAV9 and two engineered AAV vectors (PHP.eB and CAP-B10) that are noteworthy for maximizing blood-brain barrier transport were compared. CAP-B10 shares a modification in the 588 loop with PHP.eB, but also has a modification in the 455 loop, added with the goal of reducing off-target transduction. PET and optical imaging revealed that the additional modifications retained brain receptor affinity. In the liver, the accumulation of AAV9 and the engineered AAV capsids was similar (∼15% of the injected dose per cc and not significantly different between capsids at 21 h). However, the engineered capsids were primarily internalized by Kupffer cells rather than hepatocytes, and liver transduction was greatly reduced. PET reporter gene imaging after engineered AAV systemic injection provided a non-invasive method to monitor AAV-mediated protein expression over time. Through comparison with capsid tagging, differences between brain localization and transduction were revealed. In summary, AAV capsids bearing imaging tags and reporter gene payloads create a unique and powerful platform to assay the pharmacokinetics, cellular specificity and protein expression kinetics of AAV vectors in vivo, a key enabler for the field of gene therapy.

Structural basis of receptor usage by the engineered capsid AAV-PHP.eB.

Adeno-associated virus serotype 9 (AAV9) is a promising gene therapy vector for treating neurodegenerative diseases due to its ability to penetrate the blood-brain barrier. PHP.eB was engineered from AAV9 by insertion of a 7-amino acid peptide and point mutation of neighboring residues, thereby enhancing potency in the central nervous system. Here, we report a 2.24-Å resolution cryo-electron microscopy structure of PHP.eB, revealing conformational differences from other 7-mer insertion capsid variants. In PHP.eB, the 7-mer loop adopts a bent conformation, mediated by an interaction between engineered lysine and aspartate residues. Further, we identify PKD2 as the main AAV receptor (AAVR) domain recognizing both AAV9 and PHP.eB and find that the PHP.eB 7-mer partially destabilizes this interaction. Analysis of previously reported AAV structures together with our pull-down data demonstrate that the 7-mer topology determined by the lysine-aspartate interaction dictates AAVR binding strength. Our results suggest that PHP.eB's altered tropism may arise from both an additional interaction with LY6A and weakening of its AAVR interaction. Changing the insertion length, but not sequence, modifies PKD2 binding affinity, suggesting that a steric clash impedes AAVR binding. This research suggests improved library designs for future AAV selections to identify non-LY6A-dependent vectors and modulate AAVR interaction strength.

Adeno-Associated Virus Toolkit to Target Diverse Brain Cells.

Recombinant adeno-associated viruses (AAVs) are commonly used gene delivery vehicles for neuroscience research. They have two engineerable features: the capsid (outer protein shell) and cargo (encapsulated genome). These features can be modified to enhance cell type or tissue tropism and control transgene expression, respectively. Several engineered AAV capsids with unique tropisms have been identified, including variants with enhanced central nervous system transduction, cell type specificity, and retrograde transport in neurons. Pairing these AAVs with modern gene regulatory elements and state-of-the-art reporter, sensor, and effector cargo enables highly specific transgene expression for anatomical and functional analyses of brain cells and circuits. Here, we discuss recent advances that provide a comprehensive (capsid and cargo) AAV toolkit for genetic access to molecularly defined brain cell types.

Multiplexed Cre-dependent selection yields systemic AAVs for targeting distinct brain cell types.

Recombinant adeno-associated viruses (rAAVs) are efficient gene delivery vectors via intravenous delivery; however, natural serotypes display a finite set of tropisms. To expand their utility, we evolved AAV capsids to efficiently transduce specific cell types in adult mouse brains. Building upon our Cre-recombination-based AAV targeted evolution (CREATE) platform, we developed Multiplexed-CREATE (M-CREATE) to identify variants of interest in a given selection landscape through multiple positive and negative selection criteria. M-CREATE incorporates next-generation sequencing, synthetic library generation and a dedicated analysis pipeline. We have identified capsid variants that can transduce the central nervous system broadly, exhibit bias toward vascular cells and astrocytes, target neurons with greater specificity or cross the blood-brain barrier across diverse murine strains. Collectively, the M-CREATE methodology accelerates the discovery of capsids for use in neuroscience and gene-therapy applications.

Viral GPCR US28 can signal in response to chemokine agonists of nearly unlimited structural degeneracy.

Human cytomegalovirus has hijacked and evolved a human G-protein-coupled receptor into US28, which functions as a promiscuous chemokine 'sink' to facilitate evasion of host immune responses. To probe the molecular basis of US28's unique ligand cross-reactivity, we deep-sequenced CX3CL1 chemokine libraries selected on 'molecular casts' of the US28 active-state and find that US28 can engage thousands of distinct chemokine sequences, many of which elicit diverse signaling outcomes. The structure of a G-protein-biased CX3CL1-variant in complex with US28 revealed an entirely unique chemokine amino terminal peptide conformation and remodeled constellation of receptor-ligand interactions. Receptor signaling, however, is remarkably robust to mutational disruption of these interactions. Thus, US28 accommodates and functionally discriminates amongst highly degenerate chemokine sequences by sensing the steric bulk of the ligands, which distort both receptor extracellular loops and the walls of the ligand binding pocket to varying degrees, rather than requiring sequence-specific bonding chemistries for recognition and signaling.

Allosteric activation of the 5-HT3AB receptor by mCPBG.

The 5-HT3AB receptor contains three A and two B subunits in an A-A-B-A-B order. However, serotonin function at the 5-HT3AB receptor has been shown to depend solely on the A-A interface present in the homomeric receptor. Using mutations at sites on both the primary (E122) and complementary (Y146) faces of the B subunit, we demonstrate that meta-chlorophenyl biguanide (mCPBG), a 5-HT3 selective agonist, is capable of binding to and activating the 5-HT3AB receptor at all five subunit interfaces of the heteromer. Further, mCPBG is capable of allosterically modulating the activity of serotonin from these sites. While these five binding sites are similar enough that they conform to a monophasic dose - response relationship, we uncover subtle differences in the heteromeric binding sites. We also find that the A-A interface appears to contribute disproportionately to the efficacy of 5-HT3AB receptor activation.

The 5-HT3AB receptor shows an A3B2 stoichiometry at the plasma membrane.

The 5-HT3AB receptor is the best-characterized heteropentameric 5-HT3 receptor. Under conditions of heterologous expression, the 5-HT3AB receptor shows a single functionally resolvable population, suggesting the presence of a unique subunit stoichiometry; however, conflicting previous reports have suggested two different possible stoichiometries. Here we isolate plasma membrane sheets containing assembled receptors from individual HEK293T cells. We then determine the stoichiometry of 5-HT3AB receptors on the plasma membrane by fluorescence methods, employing meCFP- and meYFP-labeled A and B subunits. Over a wide range of cDNA transfection ratios, fluorescence intensity ratios are closest to values that correspond to a subunit ratio of A3B2. Förster resonance energy transfer (family FRET) efficiencies provide minor corrections (3-6%) to the subunit ratios and provide independent support for a predominantly A3B2 stoichiometry on the plasma membrane sheets. Twin FRET efficiencies support these data, also suggesting that the two B subunits are nonadjacent in most of the heteropentamers. The high-frequency variant HTR3B p.Y129S (c.386A>C, rs11767445), linked to psychiatric disease, also forms A3B2 receptors on the plasma membrane. The 5-HT3B Y129S, subunit incorporates in a slightly (11-14%) more efficient manner than the common variant. In general, most of the subunits reside within the cell. In contrast to the findings for the plasma membrane, the relative abundances and FRET characteristics of intracellular subunits depend strongly on the transfection ratio. The straightforward and unambiguous combination of plasma membrane-sheet isolation, fluorescence intensity ratios, and FRET is a generally promising procedure for determining stoichiometry of proteins on the plasma membrane.

A coupled array of noncovalent interactions impacts the function of the 5-HT3A serotonin receptor in an agonist-specific way.

The serotonin type 3A (5-HT(3)A) receptor is a Cys-loop (pentameric) neurotransmitter-gated ion channel found in the central and peripheral nervous systems and implicated in numerous diseases. In previous studies with the endogenous agonist serotonin, we identified two interactions critical for receptor function: a cation-π interaction with W183 in loop B (TrpB) and a hydrogen bond to E129 in loop A. Here we employ mutant cycle analyses utilizing conventional and unnatural amino acid mutagenesis to demonstrate that a third residue, D124 of loop A, forms two functionally important hydrogen bonds to the backbone of loop B. We also show that these three interactions, the cation-π interaction, the backbone hydrogen bonds, and the E129 hydrogen bond, are tightly coupled to each other, suggesting they function as a single unit. We also identify key functional differences between serotonin and the competitive partial agonist m-chlorophenyl biguanide (mCPBG) at these residues. mCPBG displays no cation-π at TrpB and extreme sensitivity to the positioning of E129, on which it is reliant for initiation of channel gating.

Polyglutamine fibrils are formed using a simple designed beta-hairpin model.

Polyglutamine repeats are found in proteins associated with many neurodegenerative diseases. These repeats are responsible for intracellular protein aggregation that resemble amyloid plaques and contain the hallmarks of cross-beta fibrillar structures. Recent work has suggested that the glutamines are involved in aggregation through two possible mechanisms: one involving only side-chain hydrogen bonding and a second involving interdigitation of the glutamines with tight van der Waal's packing (steric zipper model). We are interested in determining which interactions are particularly involved in early assembly processes and have developed a beta-hairpin model system to address this problem. Our model system is designed to stabilize a putative high-energy nucleating structure to provide a window to view early assembly processes. We have applied spectroscopy tools (circular dichroism, infrared, and dynamic light scattering) to probe the self-assembly of beta-sheet fibrils. These experiments established the conditions to study fibrillar morphology using atomic force microscopy. We show that fibrils are short with minimal lateral growth, suggesting that this may be a good model system for studying early assembly steps.