Computationally guided AAV engineering for enhanced gene delivery.

Gene delivery vehicles based on adeno-associated viruses (AAVs) are enabling increasing success in human clinical trials, and they offer the promise of treating a broad spectrum of both genetic and non-genetic disorders. However, delivery efficiency and targeting must be improved to enable safe and effective therapies. In recent years, considerable effort has been invested in creating AAV variants with improved delivery, and computational approaches have been increasingly harnessed for AAV engineering. In this review, we discuss how computationally designed AAV libraries are enabling directed evolution. Specifically, we highlight approaches that harness sequences outputted by next-generation sequencing (NGS) coupled with machine learning (ML) to generate new functional AAV capsids and related regulatory elements, pushing the frontier of what vector engineering and gene therapy may achieve.

Optimal trade-off control in machine learning-based library design, with application to adeno-associated virus (AAV) for gene therapy.

Adeno-associated viruses (AAVs) hold tremendous promise as delivery vectors for gene therapies. AAVs have been successfully engineered-for instance, for more efficient and/or cell-specific delivery to numerous tissues-by creating large, diverse starting libraries and selecting for desired properties. However, these starting libraries often contain a high proportion of variants unable to assemble or package their genomes, a prerequisite for any gene delivery goal. Here, we present and showcase a machine learning (ML) method for designing AAV peptide insertion libraries that achieve fivefold higher packaging fitness than the standard NNK library with negligible reduction in diversity. To demonstrate our ML-designed library's utility for downstream engineering goals, we show that it yields approximately 10-fold more successful variants than the NNK library after selection for infection of human brain tissue, leading to a promising glial-specific variant. Moreover, our design approach can be applied to other types of libraries for AAV and beyond.

Evolving membrane-associated accessory protein variants for improved adeno-associated virus production.

Manufacturing sufficient adeno-associated virus (AAV) to meet current and projected clinical needs is a significant hurdle to the growing gene therapy industry. The recently discovered membrane-associated accessory protein (MAAP) is encoded by an alternative open reading frame in the AAV cap gene that is found in all presently reported natural serotypes. Recent evidence has emerged supporting a functional role of MAAP in AAV egress, although the underlying mechanisms of MAAP function remain unknown. Here, we show that inactivation of MAAP from AAV2 by a single point mutation that is silent in the VP1 open reading frame (ORF) (AAV2-ΔMAAP) decreased exosome-associated and secreted vector genome production. We hypothesized that novel MAAP variants could be evolved to increase AAV production and thus subjected a library encoding over 1 × 106 MAAP protein variants to five rounds of packaging selection into the AAV2-ΔMAAP capsid. Between each successive packaging round, we observed a progressive increase in both overall titer and ratio of secreted vector genomes conferred by the bulk-selected MAAP library population. Next-generation sequencing uncovered enriched mutational features, and a resulting selected MAAP variant containing missense mutations and a frameshifted C-terminal domain increased overall GFP transgene packaging in AAV2, AAV6, and AAV9 capsids.

Effects of Molecular Size on Resolution in Charge Detection Mass Spectrometry.

Instrumental resolution of Fourier transform-charge detection mass spectrometry instruments with electrostatic ion trap detection of individual ions depends on the precision with which ion energy is determined. Energy can be selected using ion optic filters or from harmonic amplitude ratios (HARs) that provide Fellgett's advantage and eliminate the necessity of ion transmission loss to improve resolution. Unlike the ion energy-filtering method, the resolution of the HAR method increases with charge (improved S/N) and thus with mass. An analysis of the HAR method with current instrumentation indicates that higher resolution can be obtained with the HAR method than the best resolution demonstrated for instruments with energy-selective optics for ions in the low MDa range and above. However, this gain is typically unrealized because the resolution obtainable with molecular systems in this mass range is limited by sample heterogeneity. This phenomenon is illustrated with both tobacco mosaic virus (0.6-2.7 MDa) and AAV9 (3.7-4.7 MDa) samples where mass spectral resolution is limited by the sample, including salt adducts, and not by instrument resolution. Nevertheless, the ratio of full to empty AAV9 capsids and the included genome mass can be accurately obtained in a few minutes from 1× PBS buffer solution and an elution buffer containing 300+ mM nonvolatile content despite extensive adduction and lower resolution. Empty and full capsids adduct similarly indicating that salts encrust the complexes during late stages of droplet evaporation and that mass shifts can be calibrated in order to obtain accurate analyte masses even from highly salty solutions.

scAAVengr, a transcriptome-based pipeline for quantitative ranking of engineered AAVs with single-cell resolution.

Background: Adeno-associated virus (AAV)-mediated gene therapies are rapidly advancing to the clinic, and AAV engineering has resulted in vectors with increased ability to deliver therapeutic genes. Although the choice of vector is critical, quantitative comparison of AAVs, especially in large animals, remains challenging. Methods: Here, we developed an efficient single-cell AAV engineering pipeline (scAAVengr) to simultaneously quantify and rank efficiency of competing AAV vectors across all cell types in the same animal. Results: To demonstrate proof-of-concept for the scAAVengr workflow, we quantified - with cell-type resolution - the abilities of naturally occurring and newly engineered AAVs to mediate gene expression in primate retina following intravitreal injection. A top performing variant identified using this pipeline, K912, was used to deliver SaCas9 and edit the rhodopsin gene in macaque retina, resulting in editing efficiency similar to infection rates detected by the scAAVengr workflow. scAAVengr was then used to identify top-performing AAV variants in mouse brain, heart, and liver following systemic injection. Conclusions: These results validate scAAVengr as a powerful method for development of AAV vectors. Funding: This work was supported by funding from the Ford Foundation, NEI/NIH, Research to Prevent Blindness, Foundation Fighting Blindness, UPMC Immune Transplant and Therapy Center, and the Van Sloun fund for canine genetic research.

Gene therapy is an experimental approach to treating disease that involves altering faulty genes or replacing them with new, working copies. Most often, the new genetic material is delivered into cells using a modified virus that no longer causes disease, called a viral vector. Virus-mediated gene therapies are currently being explored for degenerative eye diseases, such as retinitis pigmentosa, and neurological disorders, like Alzheimer's and Parkinson's disease. A number of gene therapies have also been approved for treating some rare cancers, blood disorders and a childhood form of motor neuron disease. Despite the promise of virus-mediated gene therapy, there are significant hurdles to its widespread success. Viral vectors need to deliver enough genetic material to the right cells without triggering an immune response or causing serious side effects. Selecting an optimal vector is key to achieving this. A type of viruses called adeno-associated viruses (AAV) are prime candidates, partly because they can be easily engineered. However, accurately comparing the safety and efficacy of newly engineered AAVs is difficult, due to variation between test subjects and the labor and cost involved in careful testing. Öztürk et al. addressed this issue by developing an experimental pipeline called scAAVengr for comparing gene therapy vectors head-to-head. The process involves tagging potential AAV vectors with unique genetic barcodes, which can then be detected and quantified in individual cells using a technique called single-cell RNA sequencing. This means that when several vectors are used to infect lab-grown cells or a test animal at the same time, they can be tracked. The vectors can then be ranked on their ability to infect specific cell types and deliver useful genetic material. Using scAAVengr, Öztürk et al. compared viral vectors designed to target the light-sensitive cells of the retina, which allow animals to see. First, a set of promising viral vectors were evaluated using the scAAVengr pipeline in the eyes of marmosets and macaques, two small primates. Precise levels and locations of gene delivery were quantified. The top-performing vector was then identified and used to deliver Cas9, a genome editing tool, to primate retinas. Öztürk et al. also used scAAVengr to compare viral vectors in mice, analysing the vectors' ability to deliver their genetic cargo to the brain, heart, and liver. These experiments demonstrated that scAAVengr can be used to evaluate vectors in multiple tissues and in different organisms. In summary, this work outlines a method for identifying and precisely quantifying the performance of top-performing viral vectors for gene therapy. By aiding the selection of optimal viral vectors, the scAAVengr pipeline could help to improve the success of preclinical studies and early clinical trials testing gene therapies.

Genome-wide activation screens to increase adeno-associated virus production.

We describe a genome-wide screening strategy to identify target genes whose modulation increases the capacity of a cell to produce recombinant adeno-associated viral (AAV) vector. Specifically, a single-guide RNA (sgRNA) library for a CRISPR-based genome-wide transcriptional activation screen was inserted into an AAV vector, and iterative rounds of viral infection and rescue in HEK293 producer cells enabled the enrichment of sgRNAs targeting genes whose upregulation increased AAV production. Numerous gain-of-function targets were identified, including spindle and kinetochore associated complex subunit 2 (SKA2) and inositol 1, 4, 5-trisphosphate receptor interacting protein (ITPRIP). Furthermore, individual or combinatorial modulation of these targets in stable producer cell lines increased vector genomic replication and loading into AAV virions, resulting in up to a 3.8-fold increase in AAV manufacturing capacity. Our study offers an efficient approach to engineer viral vector producer cell lines and enhances our understanding of the roles of SKA2 and ITPRIP in AAV packaging.

Adeno-Associated Virus Vector for Central Nervous System Gene Therapy.

The past several years have witnessed significant advances in the development of therapeutic gene delivery for neurological disorders of the central nervous system (CNS). In particular, genome-wide sequencing analysis has deepened our understanding of mutations that underlie many monogenic disorders, which in turn has contributed to clinical advances involving adeno-associated virus (AAV) vector delivery of replacement genes to treat recessive disorders. Moreover, gene therapy has been further bolstered with advances in genome editing tools that allow researchers to silence, repair, and amend endogenous genes. However, despite strong preclinical and clinical progress, challenges remain, including delivery and safety. Here, we discuss advances in AAV engineering, recent developments in cargo design, and translation of these technologies towards clinical progress.

High-Throughput Discovery of Targeted, Minimally Complex Peptide Surfaces for Human Pluripotent Stem Cell Culture.

Human pluripotent stem cells harbor an unlimited capacity to generate therapeutically relevant cells for applications in regenerative medicine. However, to utilize these cells in the clinic, scalable culture systems that activate defined receptors and signaling pathways to sustain stem cell self-renewal are required; and synthetic materials offer considerable promise to meet these needs. De novo development of materials that target novel pathways has been stymied by a limited understanding of critical receptor interactions maintaining pluripotency. Here, we identify peptide agonists for the human pluripotent stem cell (hPSC) laminin receptor and pluripotency regulator, α6-integrin, through unbiased, library-based panning strategies. Biophysical characterization of adhesion suggests that identified peptides bind hPSCs through α6-integrin with sub-µM dissociation constants similar to laminin. By harnessing a high-throughput microculture platform, we developed predictive guidelines for presenting these integrin-targeting peptides alongside canonical binding motifs at optimal stoichiometries to generate nascent culture surfaces. Finally, when presented as self-assembled monolayers, predicted peptide combinations supported hPSC expansion, highlighting how unbiased screens can accelerate the discovery of targeted biomaterials.

Novel Lung Tropic Adeno-Associated Virus Capsids for Therapeutic Gene Delivery.

Efforts to identify mutations that underlie inherited genetic diseases combined with strides in the development of gene therapy vectors over the last three decades have culminated in the approval of several adeno-associated virus (AAV)-based gene therapies. Genetic diseases that manifest in the lung such as cystic fibrosis (CF) and surfactant deficiencies, however, have so far proven to be elusive targets. Early clinical trials in CF using AAV serotype 2 (AAV2) achieved safety, but not efficacy endpoints; however, importantly, these studies provided critical information on barriers that need to be surmounted to translate AAV lung gene therapy toward clinical success. Bolstered with an improved understanding of AAV biology and more clinically relevant lung models, next-generation molecular biology and bioinformatics approaches have given rise to novel AAV capsid variants that offer improvements in transduction efficiency, immunological profile, and the ability to circumvent physical barriers in the lung such as mucus. This review discusses the principal limiting barriers to clinical success in lung gene therapy and focuses on novel engineered AAV capsid variants that have been developed to overcome those challenges.

The delivery challenge: fulfilling the promise of therapeutic genome editing.

Genome editing has the potential to treat an extensive range of incurable monogenic and complex diseases. In particular, advances in sequence-specific nuclease technologies have dramatically accelerated the development of therapeutic genome editing strategies that are based on either the knockout of disease-causing genes or the repair of endogenous mutated genes. These technologies are progressing into human clinical trials. However, challenges remain before the therapeutic potential of genome editing can be fully realized. Delivery technologies that have serendipitously been developed over the past couple decades in the protein and nucleic acid delivery fields have been crucial to genome editing success to date, including adeno-associated viral and lentiviral vectors for gene therapy and lipid nanoparticle and other non-viral vectors for nucleic acid and protein delivery. However, the efficiency and tissue targeting capabilities of these vehicles must be further improved. In addition, the genome editing enzymes themselves need to be optimized, and challenges regarding their editing efficiency, specificity and immunogenicity must be addressed. Emerging protein engineering and synthetic chemistry approaches can offer solutions and enable the development of safe and efficacious clinical genome editing.

In vivo-directed evolution of adeno-associated virus in the primate retina.

Efficient adeno-associated virus-mediated (AAV-mediated) gene delivery remains a significant obstacle to effective retinal gene therapies. Here, we apply directed evolution - guided by deep sequencing and followed by direct in vivo secondary selection of high-performing vectors with a GFP-barcoded library - to create AAV viral capsids with the capability to deliver genes to the outer retina in primates. A replication-incompetent library, produced via providing rep in trans, was created to mitigate risk of AAV propagation. Six rounds of in vivo selection with this library in primates - involving intravitreal library administration, recovery of genomes from outer retina, and extensive next-generation sequencing of each round - resulted in vectors with redirected tropism to the outer retina and increased gene delivery efficiency to retinal cells. These viral vectors expand the toolbox of vectors available for primate retina, and they may enable less invasive delivery of therapeutic genes to patients, potentially offering retina-wide infection at a similar dosage to vectors currently in clinical use.

AAVR-Displaying Interfaces: Serotype-Independent Adeno-Associated Virus Capture and Local Delivery Systems.

Interfacing gene delivery vehicles with biomaterials has the potential to play a key role in diversifying gene transfer capabilities, including localized, patterned, and controlled delivery. However, strategies for modifying biomaterials to interact with delivery vectors must be redesigned whenever new delivery vehicles and applications are explored. We have developed a vector-independent biomaterial platform capable of interacting with various adeno-associated viral (AAV) serotypes. A water-soluble, cysteine-tagged, recombinant protein version of the recently discovered multi-AAV serotype receptor (AAVR), referred to as cys-AAVR, was conjugated to maleimide-displaying polycaprolactone (PCL) materials using click chemistry. The resulting cys-AAVR-PCL system bound to a broad range of therapeutically relevant AAV serotypes, thereby providing a platform capable of modulating the delivery of all AAV serotypes. Intramuscular injection of cys-AAVR-PCL microspheres with bound AAV vectors resulted in localized and sustained gene delivery as well as reduced spread to off-target organs compared to a vector solution. This cys-AAVR-PCL system is thus an effective approach for biomaterial-based AAV gene delivery for a broad range of therapeutic applications.

CRISPR-Cas9-Mediated Genome Editing Increases Lifespan and Improves Motor Deficits in a Huntington's Disease Mouse Model.

Huntington's disease (HD) is a currently incurable and, ultimately, fatal neurodegenerative disorder caused by a CAG trinucleotide repeat expansion within exon 1 of the huntingtin (HTT) gene, which results in the production of a mutant protein that forms inclusions and selectively destroys neurons in the striatum and other adjacent structures. The RNA-guided Cas9 endonuclease from CRISPR-Cas9 systems is a versatile technology for inducing DNA double-strand breaks that can stimulate the introduction of frameshift-inducing mutations and permanently disable mutant gene function. Here, we show that the Cas9 nuclease from Staphylococcus aureus, a small Cas9 ortholog that can be packaged alongside a single guide RNA into a single adeno-associated virus (AAV) vector, can be used to disrupt the expression of the mutant HTT gene in the R6/2 mouse model of HD following its in vivo delivery to the striatum. Specifically, we found that CRISPR-Cas9-mediated disruption of the mutant HTT gene resulted in a ∼50% decrease in neuronal inclusions and significantly improved lifespan and certain motor deficits. These results thus illustrate the potential for CRISPR-Cas9 technology to treat HD and other autosomal dominant neurodegenerative disorders caused by a trinucleotide repeat expansion via in vivo genome editing.

CRISPR-READI: Efficient Generation of Knockin Mice by CRISPR RNP Electroporation and AAV Donor Infection.

Genetically engineered mouse models harboring large sequence insertions or modifications are critical for a wide range of applications including endogenous gene tagging, conditional knockout, site-specific transgene insertion, and gene replacement; however, existing methods to generate such animals remain laborious and costly. To address this, we developed an approach called CRISPR-READI (CRISPR RNP electroporation and AAV donor infection), combining adeno-associated virus (AAV)-mediated HDR donor delivery with Cas9/sgRNA RNP electroporation to engineer large site-specific modifications in the mouse genome with high efficiency and throughput. We successfully targeted a 774 bp fluorescent reporter, a 2.1 kb CreERT2 driver, and a 3.3 kb expression cassette into endogenous loci in both embryos and live mice. CRISPR-READI is applicable to most widely used knockin schemes requiring donor lengths within the 4.9 kb AAV packaging capacity. Altogether, CRISPR-READI is an efficient, high-throughput, microinjection-free approach for sophisticated mouse genome engineering with potential applications in other mammalian species.

Spatiomechanical Modulation of EphB4-Ephrin-B2 Signaling in Neural Stem Cell Differentiation.

Interactions between EphB4 receptor tyrosine kinases and their membrane-bound ephrin-B2 ligands on apposed cells play a regulatory role in neural stem cell differentiation. With both receptor and ligand constrained to move within the membranes of their respective cells, this signaling system inevitably experiences spatial confinement and mechanical forces in conjunction with receptor-ligand binding. In this study, we reconstitute the EphB4-ephrin-B2 juxtacrine signaling geometry using a supported-lipid-bilayer system presenting laterally mobile and monomeric ephrin-B2 ligands to live neural stem cells. This experimental platform successfully reconstitutes EphB4-ephrin-B2 binding, lateral clustering, downstream signaling activation, and neuronal differentiation, all in a configuration that preserves the spatiomechanical aspects of the natural juxtacrine signaling geometry. Additionally, the supported bilayer system allows control of lateral movement and clustering of the receptor-ligand complexes through patterns of physical barriers to lateral diffusion fabricated onto the underlying substrate. The results from this study reveal a distinct spatiomechanical effect on the ability of EphB4-ephrin-B2 signaling to induce neuronal differentiation. These observations parallel similar studies of the EphA2-ephrin-A1 system in a very different biological context, suggesting that such spatiomechanical regulation may be a common feature of Eph-ephrin signaling.

hPSC-Derived Striatal Cells Generated Using a Scalable 3D Hydrogel Promote Recovery in a Huntington Disease Mouse Model.

Huntington disease (HD) is an inherited, progressive neurological disorder characterized by degenerating striatal medium spiny neurons (MSNs). One promising approach for treating HD is cell replacement therapy, where lost cells are replaced by MSN progenitors derived from human pluripotent stem cells (hPSCs). While there has been remarkable progress in generating hPSC-derived MSNs, current production methods rely on two-dimensional culture systems that can include poorly defined components, limit scalability, and yield differing preclinical results. To facilitate clinical translation, here, we generated striatal progenitors from hPSCs within a fully defined and scalable PNIPAAm-PEG three-dimensional (3D) hydrogel. Transplantation of 3D-derived striatal progenitors into a transgenic mouse model of HD slowed disease progression, improved motor coordination, and increased survival. In addition, the transplanted cells developed an MSN-like phenotype and formed synaptic connections with host cells. Our results illustrate the potential of scalable 3D biomaterials for generating striatal progenitors for HD cell therapy.

Engineered viral vectors for functional interrogation, deconvolution, and manipulation of neural circuits.

Optimization of traditional replication-competent viral tracers has granted access to immediate synaptic partners of target neuronal populations, enabling the dissection of complex brain circuits into functional neural pathways. The excessive virulence of most conventional tracers, however, impedes their utility in revealing and genetically perturbing cellular function on long time scales. As a promising alternative, the natural capacity of adeno-associated viral (AAV) vectors to safely mediate persistent and robust gene expression has stimulated strong interest in adapting them for sparse neuronal labeling and physiological studies. Furthermore, increasingly refined engineering strategies have yielded novel AAV variants with enhanced target specificity, transduction, and retrograde trafficking in the CNS. These potent vectors offer new opportunities for characterizing the identity and connectivity of single neurons within immense networks and modulating their activity via robust delivery of functional genetic tools.

Adeno-associated virus-mediated delivery of CRISPR-Cas9 for genome editing in the central nervous system.

The emergence of CRISPR-Cas9 as a powerful genome editing tool has led to several studies exploring its potential to treat neurological disorders. Cas9 and its sgRNA can be readily engineered to target any gene and can be multiplexed to target several genes at once. Furthermore, the use of adeno-associated virus (AAV) to deliver with Cas9 and its sgRNA is a promising therapeutic combination with strong potential to reach the clinic. Here we discuss how Cas9 editing has been utilized for gene insertion, knockout, and deletion in vivo for applications in the central nervous system (CNS). Furthermore, we highlight major challenges that remain for AAV-Cas9-sgRNA clinical translation.

Screening for Neutralizing Antibodies Against Natural and Engineered AAV Capsids in Nonhuman Primate Retinas.

Adeno-associated virus (AAV) has shown promise as a therapeutic gene delivery vector for inherited retinal degenerations in both preclinical disease models and human clinical trials. The retinas of nonhuman primates (NHPs) share many anatomical similarities to humans and are an important model for evaluating AAV gene delivery. Recent evidence has shown that preexisting immunity in the form of neutralizing antibodies (NABs) in NHPs strongly correlates with weak or lack of AAV transduction in the retina when administered intravitreally, work with translational implications. This necessitates prescreening of NHPs before intravitreal delivery of AAV. In this chapter, we describe a method for screening NHP serum for preexisting NABs.

In Vivo Selection of a Computationally Designed SCHEMA AAV Library Yields a Novel Variant for Infection of Adult Neural Stem Cells in the SVZ.

Directed evolution continues to expand the capabilities of complex biomolecules for a range of applications, such as adeno-associated virus vectors for gene therapy; however, advances in library design and selection strategies are key to develop variants that overcome barriers to clinical translation. To address this need, we applied structure-guided SCHEMA recombination of the multimeric adeno-associated virus (AAV) capsid to generate a highly diversified chimeric library with minimal structural disruption. A stringent in vivo Cre-dependent selection strategy was implemented to identify variants that transduce adult neural stem cells (NSCs) in the subventricular zone. A novel variant, SCH9, infected 60% of NSCs and mediated 24-fold higher GFP expression and a 12-fold greater transduction volume than AAV9. SCH9 utilizes both galactose and heparan sulfate as cell surface receptors and exhibits increased resistance to neutralizing antibodies. These results establish the SCHEMA library as a valuable tool for directed evolution and SCH9 as an effective gene delivery vector to investigate subventricular NSCs.