Synthetic Adeno-associated Virus Research Articles

Combined AAV-mediated gene replacement therapy improves auditory function in a mouse model of human DFNB42 deafness

Hearing loss is a common disorder affecting nearly 20% of the world's population. Recently, studies have shown that inner ear gene therapy can improve auditory function in several mouse models of hereditary hearing loss. In most of these studies, the underlying mutations affect only a small number of cell types of the inner ear (e.g., sensory hair cells). Here, we applied inner ear gene therapy to the Ildr1Gt(D178D03)Wrst (Ildr1w-/-) mouse, a model of human DFNB42, non-syndromic autosomal recessive hereditary hearing loss associated with ILDR1 variants. ILDR1 is an integral protein of the tricellular tight junction complex and is expressed by diverse inner ear cell types in the organ of Corti and the cochlear lateral wall. We simultaneously applied two synthetic adeno-associated viruses (AAVs) with different tropism to deliver Ildr1 cDNA to the Ildr1w-/- mouse inner ear: one targeting the organ of Corti (AAV2.7m8) and the other targeting the cochlear lateral wall (AAV8BP2). We showed that combined AAV2.7m8/AAV8BP2 gene therapy improves cochlear structural integrity and auditory function in Ildr1w-/- mice.

Progress in Bioengineering of Myotropic Adeno-Associated Viral Gene Therapy Vectors

The ability to specifically, safely, and efficiently transfer therapeutic payloads to the striated musculature via a minimally invasive delivery route remains one of the most important but also most ambitious aims in human gene therapy. Over the past two decades, a flurry of groups have harnessed recombinant adeno-associated viruses (AAVs) for this purpose, carrying cargoes that were packaged either in one of the various wild-type capsids or in a synthetic protein shell derived by molecular bioengineering. In this study, we provide an overview over the most commonly used techniques for the enrichment of muscle-specific (myotropic) AAV capsids, typically starting off with the genetic diversification of one or more extant wild-type sequences, followed by the stratification of the ensuing capsid libraries in different muscle types in small or large animals. These techniques include the shuffling of multiple parental capsid genes, peptide display in exposed capsid loops, mutagenesis of individual capsid residues, creation of chimeras between two viral parents, or combinations thereof. Moreover, we highlight alternative experimental or bioinformatic strategies such as ancestral reconstruction or rational design, all of which have already been employed successfully to derive synthetic AAV capsids or vectors with unprecedented in vivo efficiency and/or specificity in the musculature. Most recently, these efforts have culminated in the isolation of unique clades of myotropic vectors called AAVMYO or MyoAAV that have in common the display of the amino acid motif RGD (arginine-glycine-aspartate) on the capsid surface and that exhibit the highest transduction rate in striated muscles of mice or nonhuman primates reported to date. Finally, we note essential looming improvements that will facilitate and accelerate clinical translation of these latest generations of myotropic AAVs, including the identification and utilization of capsid selection or validation schemes that promise optimal translation in humans, and continued efforts to enhance patient safety by minimizing hepatic off-targeting.

An evolved AAV variant enables efficient genetic engineering of murine T cells

Precise targeting of large transgenes to Tcells using homology-directed repair has been transformative for adoptive cell therapies and Tcell biology. Delivery of DNA templates via adeno-associated virus (AAV) has greatly improved knockin efficiencies, but the tropism of current AAV serotypes restricts their use to human Tcells employed in immunodeficient mouse models. To enable targeted knockins in murine Tcells, we evolved Ark313, a synthetic AAV that exhibits high transduction efficiency in murine Tcells. We performed a genome-wide knockout screen and identified QA2 as an essential factor for Ark313 infection. We demonstrate that Ark313 can be used for nucleofection-free DNA delivery, CRISPR-Cas9-mediated knockouts, and targeted integration of large transgenes. Ark313 enables preclinical modeling of Trac-targeted CAR-T and transgenic TCR-T cells in immunocompetent models. Efficient gene targeting in murine Tcells holds great potential for improved cell therapies and opens avenues in experimental Tcell immunology.

Identification of adeno-associated virus variants for gene transfer into human neural cell types by parallel capsid screening

Human brain cells generated by in vitro cell programming provide exciting prospects for disease modeling, drug discovery and cell therapy. These applications frequently require efficient and clinically compliant tools for genetic modification of the cells. Recombinant adeno-associated viruses (AAVs) fulfill these prerequisites for a number of reasons, including the availability of a myriad of AAV capsid variants with distinct cell type specificity (also called tropism). Here, we harnessed a customizable parallel screening approach to assess a panel of natural or synthetic AAV capsid variants for their efficacy in lineage-related human neural cell types. We identified common lead candidates suited for the transduction of directly converted, early-stage induced neural stem cells (iNSCs), induced pluripotent stem cell (iPSC)-derived later-stage, radial glia-like neural progenitors, as well as differentiated astrocytic and mixed neuroglial cultures. We then selected a subset of these candidates for functional validation in iNSCs and iPSC-derived astrocytes, using shRNA-induced downregulation of the citrate transporter SLC25A1 and overexpression of the transcription factor NGN2 for proofs-of-concept. Our study provides a comparative overview of the susceptibility of different human cell programming-derived brain cell types to AAV transduction and a critical discussion of the assets and limitations of this specific AAV capsid screening approach.

Overloaded Adeno-Associated Virus as a Novel Gene Therapeutic Tool for Otoferlin-Related Deafness.

Hearing impairment is the most common sensory disorder in humans. So far, rehabilitation of profoundly deaf subjects relies on direct stimulation of the auditory nerve through cochlear implants. However, in some forms of genetic hearing impairment, the organ of Corti is structurally intact and therapeutic replacement of the mutated gene could potentially restore near natural hearing. In the case of defects of the otoferlin gene (OTOF), such gene therapy is hindered by the size of the coding sequence (~6 kb) exceeding the cargo capacity (<5 kb) of the preferred viral vector, adeno-associated virus (AAV). Recently, a dual-AAV approach was used to partially restore hearing in deaf otoferlin knock-out (Otof-KO) mice. Here, we employed in vitro and in vivo approaches to assess the gene-therapeutic potential of naturally-occurring and newly-developed synthetic AAVs overloaded with the full-length Otof coding sequence. Upon early postnatal injection into the cochlea of Otof-KO mice, overloaded AAVs drove specific expression of otoferlin in ~30% of all IHCs, as demonstrated by immunofluorescence labeling and polymerase chain reaction. Recordings of auditory brainstem responses and a behavioral assay demonstrated partial restoration of hearing. Together, our results suggest that viral gene therapy of DFNB9—using a single overloaded AAV vector—is indeed feasible, reducing the complexity of gene transfer compared to dual-AAV approaches.

Production, Processing, and Characterization of Synthetic AAV Gene Therapy Vectors.

Over the last two decades, gene therapy vectors based on wild-type Adeno-associated viruses (AAV) are safe and efficacious in numerous clinical trials and are translated into three approved gene therapy products. Concomitantly, a large body of preclinical work has illustrated the power and potential of engineered synthetic AAV capsids that often excel in terms of an organ or cell specificity, the efficiency of in vitro or in vivo gene transfer, and/or reactivity with anti-AAV immune responses. In turn, this has created a demand for new, scalable, easy-to-implement, and plug-and-play platform processes that are compatible with the rapidly increasing range of AAV capsid variants. Here, the focus is on recent advances in methodologies for downstream processing and characterization of natural or synthetic AAV vectors, comprising different chromatography techniques and thermostability measurements. To illustrate the breadth of this portfolio, two chimeric capsids are used as representative examples that are derived through forward- or backwards-directed molecular evolution, namely, AAV-DJ and Anc80. Collectively, this ever-expanding arsenal of technologies promises to facilitate the development of the next AAV vector generation derived from synthetic capsids and to accelerate their manufacturing, and to thus boost the field of human gene therapy.

Co-Prevalence of Pre-Existing Immunity to Different Serotypes of Adeno-Associated Virus (AAV) in Adults with Hemophilia

Background: Recombinant and synthetic adeno-associated virus (AAV) vectors are in development for gene transfer in patients with hemophilia A (HA) or hemophilia B (HB). These include liver-directed recombinant AAV8 vectors BAX 888/SHP654/TAK-754 factor VIII (FVIII) gene therapy (GT) for severe HA, and SHP648/TAK-748 factor IX (FIX) GT for HB (Baxalta US Inc., a Takeda company, Lexington, MA, USA). However, environmental exposure to wild-type AAVs can result in individuals developing antibodies and cell-mediated immune responses to the naturally occurring AAV. While natural exposure to AAV does not result in any known disease in humans, presence of preexisting immunity can block delivery and prevent sustained expression of the transgene by an AAV-based vector in a gene therapy setting. Of the AAV serotypes, AAV2 is the most frequently encountered natural human infection and AAV5 and AAV8 have been the most commonly used vectors for hemophilia GT. Therefore, it is important to assess the prevalence and co-prevalence of antibody and T cell-mediated responses against each of these AAV serotypes and to better characterize the association between humoral and cellular immunity in people with hemophilia. Aims: To determine the prevalence of preexisting antibody-mediated immunity against AAV2, AAV5 and AAV8 and the association between AAV8-specific humoral and cell-mediated responses in adult patients with HA and HB in an international prospective, epidemiological study. Methods: This ongoing seroprevalence study involved adult male patients (18-75 years of age) with severe HA (&lt;1% plasma FVIII activity) or severe/moderate HB (≤2% plasma FIX activity) recruited from hemophilia treatment centers in the United States and Europe (NCT03185897). Participants consented to collection of peripheral blood at either a single or multiple annual outpatient study visits, in order to explore fluctuations of the immune response over time. Local ethics committee approval was obtained. Titers for anti-AAV2, anti-AAV5 and anti-AAV8 neutralizing antibodies (NAbs) were determined using a cell-based transduction inhibition assay, with seropositivity defined as a titer ≥1:5. Titers for anti-AAV2, anti-AAV5 and anti-AAV8 binding antibodies (BAbs) were quantitated by indirect enzyme-linked immunosorbent assay (ELISA), with seropositivity defined as a titer ≥1:80. Cell-mediated immune responses to AAV8 peptide antigens were measured in peripheral blood mononuclear cells using an interferon-γ enzyme-linked immunospot (ELISpot) assay. Samples with a signal ≥3 times background and &gt;60 spots per million cells were defined as positive. Results: Here we present data from patients who completed at least a single visit at the time of the interim, one year data cut (November 9, 2018). Of 242 patients enrolled (mean ± SD age: 35.3 ± 11.4 years), 194 patients had HA and 48 patients had HB. The overall co-prevalence of NAbs and BAbs to AAV2, AAV5 and AAV8 was 39.7% (HA: 38.1%, 72/189; HB: 45.8%, 22/48) and 16.1% (HA: 16.5%, 31/188; HB: 14.6%, 7/48) respectively, with further details shown in Table 1. Overall, 38.3% of patients (82/214) exhibited a T cell-mediated immune response to AAV8 peptide antigens (HA: 35.9%, 61/170; HB: 47.7%, 21/44). Among patients with AAV-8-specific NAbs, 37.9% (39/103) demonstrated positive AAV8-specific ELISPOT results. (HA: 35.7%, 30/84; HB: 47.4%, 9/19). Conclusion: The findings from this ongoing study demonstrate that approximately 50% of patients with hemophilia have preexistent NAb responses to AAV2, AAV5 or AAV8 with 40% demonstrating co-prevalence to all 3 evaluated AAV serotypes. Similar percentages of patients exhibited a positive cellular response to AAV8 antigens. Further, patients with HB demonstrated a slightly higher co-prevalence and a higher cellular response than patients with HA. In the combined HA and HB cohorts, co-prevalence was almost 40% for AAV8-specific humoral and T-cell mediated immunity. These data will add to our appreciation of preexisting AAV immunity that prevent patient participation in gene therapy trials. Disclosures Rajavel: Baxalta US Inc., a Takeda company: Employment, Equity Ownership. Ayash-Rashkovsky:Baxalta US Inc., a Takeda company: Employment, Equity Ownership. Tang:Baxalta US Inc., a Takeda company: Employment, Equity Ownership. Gangadharan:Baxalta Innovations GmbH, a Takeda company: Employment. de la Rosa:Baxalta Innovations GmbH, a Takeda company: Employment. Ewenstein:Baxalta US Inc., a Takeda company: Employment, Equity Ownership, Other: a Takeda stock owner.

AAV2.7m8 is a powerful viral vector for inner ear gene therapy

Adeno-associated virus (AAV) has been successfully used to deliver gene therapy to improve auditory function in mouse models of hereditary hearing loss. Many forms of hereditary hearing loss have mutations which affect the cochlear hair cells, the mechanosensory cells which allow for sound detection and processing. While most conventional AAVs infect inner hair cells (IHCs) with various efficiencies, they infect outer hair cells (OHCs) and supporting cells at lower levels in the cochlea. Here we examine the infection patterns of two synthetic AAVs (AAV2.7m8 and AAV8BP2) in the mouse inner ear. AAV2.7m8 infects both IHCs and OHCs with high efficiency. In addition, AAV2.7m8 infects inner pillar cells and inner phalangeal cells with high efficiency. Our results suggest that AAV2.7m8 is an excellent viral vector for inner ear gene therapy targeting cochlear hair cells and supporting cells, and it will likely greatly expand the potential applications for inner ear gene therapy.

Efficient Gene Transfer to Kidney Mesenchymal Cells Using a Synthetic Adeno-Associated Viral Vector.

After injury, mesenchymal progenitors in the kidney interstitium differentiate into myofibroblasts, cells that have a critical role in kidney fibrogenesis. The ability to deliver genetic material to myofibroblast progenitors could allow new therapeutic approaches to treat kidney fibrosis. Preclinical and clinical studies show that adeno-associated viruses (AAVs) efficiently and safely transduce various tissue targets in vivo; however, protocols for transduction of kidney mesenchymal cells have not been established. We evaluated the transduction profiles of various pseudotyped AAV vectors expressing either GFP or Cre recombinase reporters in mouse kidney and human kidney organoids. Of the six AAVs tested, a synthetic AAV called Anc80 showed specific and high-efficiency transduction of kidney stroma and mesangial cells. We characterized the cell specificity, dose dependence, and expression kinetics and showed the efficacy of this approach by knocking out Gli2 from kidney mesenchymal cells by injection of Anc80-Cre virus into either homozygous or heterozygous Gli2-floxed mice. After unilateral ureteral obstruction, the homozygous Gli2-floxed mice had less fibrosis than the Gli2 heterozygotes had. We observed the same antifibrotic effect in β-catenin-floxed mice injected with Anc80-Cre virus before obstructive injury, strongly supporting a central role for canonical Wnt signaling in kidney myofibroblast activation. Finally, we showed that the Anc80 synthetic virus can transduce the mesenchymal lineage in human kidney organoids. These studies establish a novel method for inducible knockout of floxed genes in mouse mesangium, pericytes, and perivascular fibroblasts and are the foundation for future gene therapy approaches to treat kidney fibrosis.

Small But Increasingly Mighty: Latest Advances in AAV Vector Research, Design, and Evolution.

Recombinant gene delivery vectors derived from naturally occurring or genetically engineered adeno-associated viruses (AAV) have taken center stage in human gene therapy, fueled by rapidly accumulating and highly encouraging clinical data. Nonetheless, it has also become evident that the current generation of AAV vectors will require improvements in transduction potency, antibody evasion, and cell specificity in order to realize their full potential and to widen applicability in larger patient cohorts. Fortunately, in the recent past, the field has seen a flurry of exciting new developments that enhance our understanding of AAV vector biology, including virus-host interactions, and/or that expand our arsenal of technologies for AAV capsid design and evolution. This review highlights a collection of latest advances in these areas, which, in the authors' opinion, hold particular promise to propel the AAV vector field forward in the near future, especially when applied in combination. These include fundamental novel insights into the AAV life cycle, from an unexpected role of autophagy and interactions with other viruses to the (re-)discovery of a universal AAV receptor and the function of AAV-AAP for capsid assembly. Concurrently, recent successes in the rational design of next-generation synthetic AAV capsids are pointed out, exemplified by the structure-guided derivation of AAV mutants displaying robust in vivo immune evasion. Finally, a variety of new and innovative strategies for high-throughput generation and screening of AAV capsid libraries are briefly reviewed, including Cre recombinase-based selection, ancestral AAV capsid reconstruction, and DNA barcoding of AAV genomes. All of these examples showcase the present momentum in the AAV field and, together with work by many other academic or industrial entities, raise substantial optimism that the remaining hurdles for human gene therapy with AAV vectors will (soon) be overcome.

Next-generation AAV vectors for clinical use: an ever-accelerating race

During the past five decades, it has become evident that Adeno-associated virus (AAV) represents one of the most potent, most versatile, and thus most auspicious platforms available for gene delivery into cells, animals and, ultimately, humans. Particularly attractive is the ease with which the viral capsid-the major determinant of virus-host interaction including cell specificity and antibody recognition-can be modified and optimized at will. This has motivated countless researchers to develop high-throughput technologies in which genetically engineered AAV capsid libraries are subjected to a vastly hastened emulation of natural evolution, with the aim to enrich novel synthetic AAV capsids displaying superior features for clinical application. While the power and potential of these forward genetics approaches is undisputed, they are also inherently challenging as success depends on a combination of library quality, fidelity, and complexity. Here, we will describe and discuss two original, very exciting strategies that have emerged over the last three years and that promise to alleviate at least some of these concerns, namely, (i) a reverse genetics approach termed "ancestral AAV sequence reconstruction," and (ii) AAV genome barcoding as a technology that can advance both, forward and reverse genetics stratagems. Notably, despite the conceptual differences of these two technologies, they pursue the same goal which is tailored acceleration of AAV evolution and thus winning the race for the next-generation AAV vectors for clinical use.

A synthetic AAV vector enables safe and efficient gene transfer to the mammalian inner ear.

Efforts to develop gene therapies for hearing loss have been hampered by the lack of safe, efficient, and clinically relevant delivery modalities1, 2. Here we demonstrate the safety and efficiency of Anc80L65, a rationally designed synthetic vector3, for transgene delivery to the mouse cochlea. Cochlear explants incubated with Anc80L65 encoding eGFP demonstrated high level transduction of inner and outer hair cells (60–100%). Injection of Anc80L65 through the round window membrane resulted in highly efficient transduction of inner and outer hair cells, a substantial improvement over conventional adeno-associated virus (AAV) vectors. Anc80L65 round window injection was well tolerated, as indicated by sensory cell function, hearing and vestibular function, and immunologic parameters. The ability of Anc80L65 to target outer hair cells at high rates, a requirement for restoration of complex auditory function, may enable future gene therapies for hearing and balance disorders.