T-cell specific in vivo gene delivery with DART-AAVs targeted to CD8.

One of the biggest challenges for in vivo gene therapy are vectors mediating highly selective gene transfer into a defined population of therapy-relevant cells. Here we present DARPin-targeted AAVs (DART-AAVs) displaying DARPins specific for human and murine CD8. Insertion of DARPins into the GH2/GH3 loop of the capsid protein 1 (VP1) of AAV2 and AAV6 resulted in high selectivity for CD8-positive T cells with unimpaired gene delivery activity. Remarkably, the capsid core structure was unaltered with protruding DARPins detectable. In complex primary cell mixtures, including donor blood or systemic injections into mice, the CD8-targeted AAVs were by far superior to unmodified AAV2 and AAV6 in terms of selectivity, target cell viability, and gene transfer rates. In vivo, up to 80% of activated CD8+ T cells were hit upon a single vector injection into conditioned humanized or immunocompetent mice. While gene transfer rates decreased significantly under non-activated conditions, genomic modification selectively in CD8+ T cells was still detectable upon Cre delivery into indicator mice. In both mouse models, selectivity for CD8+ T cells was close to absolute with exceptional detargeting from liver. The CD8-AAVs described here expand strategies for immunological research and in vivo gene therapy options.

Temporal restriction of Cas9 expression improves CRISPR-mediated deletion efficacy and fidelity.

Clinical application of CRISPR-Cas9 technology for large deletions of somatic mutations is inefficient, and methods to improve utility suffer from our inability to rapidly assess mono- vs. biallelic deletions. Here we establish a model system for investigating allelic heterogeneity at the single-cell level and identify indel scarring from non-simultaneous nuclease activity at gRNA cut sites as a major barrier to CRISPR-del efficacy both in vitro and in vivo. We show that non-simultaneous nuclease activity is partially prevented via restriction of CRISPR-Cas9 expression via inducible adeno-associated viruses (AAVs) or lipid nanoparticles (LNPs). Inducible AAV-based expression of CRISPR-del machinery significantly improved mono- and biallelic deletion frequency in vivo, supporting the use of the Xon cassette over traditional constitutively expressing AAV approaches. These data depicting improvements to deletions and insight into allelic heterogeneity after CRISPR-del will inform therapeutic approaches for phenotypes that require either large mono- or biallelic deletions, such as autosomal recessive diseases or where mutant allele-specific gRNAs are not readily available, or in situations where the targeted sequence for excision is located multiple times in a genome.

Profiling Mouse Brain Single-Cell-Type Proteomes Via Adeno-Associated Virus-Mediated Proximity Labeling and Mass Spectrometry.

Single-cell-type proteomics is an emerging field of research that combines cell-type specificity with the comprehensive proteome coverage offered by bulk proteomics. However, the extraction of single-cell-type proteomes remains a challenge, particularly for hard-to-isolate cells like neurons. In this chapter, we present an innovative technique for profiling single-cell-type proteomes using adeno-associated virus (AAV)-mediated proximity labeling (PL) and tandem-mass-tag (TMT) mass spectrometry. This technique eliminates the need for cell isolation and offers a streamlined workflow, including AAV delivery to express TurboID (an engineered biotin ligase) controlled by cell-type-specific promoters, biotinylated protein purification, on-bead digestion, TMT labeling, and liquid chromatography-mass spectrometry (LC-MS). We examined this method by analyzing distinct brain cell types in mice. Initially, recombinant AAVs were used to concurrently express TurboID and mCherry proteins driven by neuron- or astrocyte-specific promoters, which was validated through co-immunostaining with cellular markers. With biotin purification and TMT analysis, we successfully identified around 10,000 unique proteins from a few micrograms of protein samples with high reproducibility. Our statistical analyses revealed that these proteomes encompass cell-type-specific cellular pathways. By utilizing this technique, researchers can explore the proteomic landscape of specific cell types, paving the way for new insights into cellular processes, deciphering disease mechanisms, and identifying therapeutic targets in neuroscience and beyond.

Fast and efficient size exclusion chromatography of adeno associated viral vectors with 2.5 micrometer particle low adsorption columns.

More and more transformative gene therapies (GTx) are reaching commercialization stage and many of them use Adeno Associated Viruses (AAVs) as their vector. Being larger than therapeutic antibodies, their size variant analysis poses an analytical challenge that must be addressed to speed up the development processes. Size exclusion chromatography (SEC) can provide critical information on the quality and purity of the product, but its full potential is not yet utilized by currently applied columns that are (i) packed with relatively large particles, (ii) prepared exclusively in large formats and (iii) built using metal hardware that is prone to secondary interactions. In this paper, we investigate the use of state-of-the-art sub-3 µm particles to address existing limitations. A prototype 2.5 µm column was found to deliver superior kinetic efficiency, significant reduction in run times and increased resolution of separations. No evidence for shear or sample sieving effects were found during comparisons with conventional 5 µm columns. Moreover, use of low adsorption hardware enabled the application of a wide range of mobile phase conditions and a chance to apply a more robust platform method for several AAV serotypes. The resulting method was tested for its reproducibility as well as utility for critical quality attribute assays, including multiangle light scattering based (MALS) measurements of size and molar mass. Thus, a new tool for higher resolution, higher throughput size variant analysis of AAVs has been described.

Generation of hepatitis C virus-resistant liver cells by genome editing-mediated stable expression of RNA aptamer.

Hepatitis C virus (HCV) infections frequently recur after liver transplantation in patients with HCV-related liver diseases. Approximately 30% of these patients progress to cirrhosis within 5 years after surgery. In this study, we proposed an effective therapeutic strategy to overcome the recurrence of HCV. CRISPR-Cas9 was used to insert an expression cassette encoding an RNA aptamer targeting HCV NS5B replicase as an anti-HCV agent into adeno-associated virus integration site 1 (AAVS1), known as a "safe harbor," in a hepatocellular carcinoma cell line to confer resistance to HCV. The RNA aptamer expression system based on a dihydrofolate reductase minigene was precisely knocked in into AAVS1, leading to the stable expression of aptamer RNA in the developed cell line. HCV replication was effectively inhibited at both the RNA and protein levels in cells transfected with HCV RNA or infected with HCV. RNA immunoprecipitation and competition experiments strongly suggested that this HCV inhibition was due to the RNA aptamer-mediated sequestration of HCV NS5B. No off-target insertion of the RNA aptamer expression construct was observed. The findings suggest that HCV-resistant liver cells produced by genome editing technology could be used as a new alternative in the development of a treatment for HCV-induced liver diseases.

[Rapamycin mediated caspase 9 homodimerization to safeguard human pluripotent stem cell therapy].

Human induced pluripotent stem cells (hiPSCs) are promising in regenerative medicine. However, the pluripotent stem cells (PSCs) may form clumps of cancerous tissue, which is a major safety concern in PSCs therapies. Rapamycin is a safe and widely used immunosuppressive pharmaceutical that acts through heterodimerization of the FKBP12 and FRB fragment. Here, we aimed to insert a rapamycin inducible caspase 9 (riC9) gene in a safe harbor AAVS1 site to safeguard hiPSCs therapy by drug induced homodimerization. The donor vector containing an EF1α promoter, a FRB-FKBP-Caspase 9 (CARD domain) fusion protein and a puromycin resistant gene was constructed and co-transfected with sgRNA/Cas9 vector into hiPSCs. After one to two weeks screening with puromycin, single clones were collected for genotype and phenotype analysis. Finally, rapamycin was used to induce the homodimerization of caspase 9 to activate the apoptosis of the engineered cells. After transfection of hiPSCs followed by puromycin screening, five cell clones were collected. Genome amplification and sequencing showed that the donor DNA has been precisely knocked out at the endogenous AAVS1 site. The engineered hiPSCs showed normal pluripotency and proliferative capacity. Rapamycin induced caspase 9 activation, which led to the apoptosis of all engineered hiPSCs and its differentiated cells with different sensitivity to drugs. In conclusion, we generated a rapamycin-controllable hiPSCs survival by homodimerization of caspase 9 to turn on cell apoptosis. It provides a new strategy to guarantee the safety of the hiPSCs therapy.

Cranial Window for Acute and Chronic Optical Access to Record Neuronal Network Dynamics in the Olfactory Bulb.

Cranial window implant is the preparation of choice for acute and chronic optical access to a given brain area. The cranial window provides a stable preparation, which can last for months. This window allows to follow the activity of distinct population of neurons expressing genetically encoded fluorescent reporters of activity in awake, behaving, head-fixed animals. The optical access can also be exploited for acute imaging, in anesthetized animals. Here we provide a detailed protocol for acute and chronic cranial window implantations in the olfactory bulb. We also provide the procedure to perform injections of adeno-associated viruses expressing genetically encoded fluorescent sensors in the same area.

Patient-reported outcomes in vasculitis.

Systemic vasculitis encompasses a group of multisystem disorders; both the diseases and the treatment strategies can have a significant impact on a patient's health-related quality of life (HRQoL). Using patient-reported outcome measures (PROMs) and patient-reported experience measures (PREMs) to evaluate the patient's view of their condition, treatments, and healthcare journey is essential to the patient-centered care approach. In this paper, we discuss the use of generic, disease-specific, and treatment-specific PROMs and PREMs in systemic vasculitis and future research goals.

Identification AAVS1-like locus from the porcine genome and site-specific integration of recombinase-mediated cassette exchange using CRISPR/Cas9.

Gene integration at site-specific loci is a critical approach for understanding the function of a gene in cells or animals. The AAVS1 locus is a well-known safe harbor for human and mouse studies. In this study, we found an AAVS1-like sequence (pAAVS1) in the porcine genome using the Genome Browser and designed TALEN and CRISPR/Cas9 to target the pAAVS1. The efficiency of CRISPR/Cas9 in porcine cells was superior to that of TALEN. We added a loxP-lox2272 sequences to the pAAVS1 targeting donor vector containing GFP for further exchange of various transgenes via recombinase-mediated cassette exchange (RMCE). The donor vector and CRISPR/Cas9 components were transfected into porcine fibroblasts. Targeted cells of CRISPR/Cas9-mediated homologous recombination were identified by antibiotic selection. Gene knock-in was confirmed by PCR. To induce RMCE, another donor vector containing the loxP-lox2272 and inducible Cre recombinase was cloned. The Cre-donor vector was transfected into the pAAVS1 targeted cell line, and RMCE was induced by adding doxycycline to the culture medium. RMCE in porcine fibroblasts was confirmed using PCR. In conclusion, gene targeting at the pAAVS1 and RMCE in porcine fibroblasts was successful. This technology will be useful for future porcine transgenesis studies and the generation of stable transgenic pigs.

Optimization of adeno-associated virus (AAV) gene delivery into human bone marrow stem cells (hBMSCs).

Background: Efficiently delivering nucleic acid into mammalian cells is essential to overexpress genes for assessing gene functions. Human bone marrow stem cells (hBMSCs) are the most studied tissue-derived stem cells. Adeno-associated viruses (AAVs) have been used to deliver DNA into hBMSCs for various purposes. Current literature reported that transduction efficiencies of up to 65% could be achieved by AAV gene delivery into hBMSCs. Further improvement of efficiency is needed and possible. This study tested a selection of AAV serotypes for high-efficient DNA delivery into hBMSCs. Methods: hBMSCs from different donors were infected with different serotypes of AAVs containing the enhanced green fluorescence protein (eGFP) reporter gene driven by the CMV promoter. Green fluorescence was monitored in the infected cells at five-day intervals. Cells were collected at designated time points after the infection for reverse-transcription polymerase chain reaction (RT-PCR) and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) to assess eGFP mRNA transcription. Results: The results indicated that the order of transduction efficiency of the AAV serotypes was AAV2 > AAV2.7m8 > AAV6 > AAV6.2 > AAV1 > AAV-DJ. AAV2 could achieve almost 100% transduction at the multiplicity of infection (MOI) greater than 100K. Over 90% of cells could be transduced at 20K to 50K MOI. About 80% transduction was seen at MOIs of 10K and 15K. RT-PCR analysis showed that eGFP mRNA could be detected from day 5 to day 30 post-AAV infection. The differences in the observed transduction efficiencies of the hBMSCs from different patients indicate donor-to-donor variability, and increased eGFP mRNA was generally seen after day 15 post-AAV2 infection. Maximal eGFP transcription was detected on day 30 post-infection. Conclusions: We conclude that AAV2 and AAV2.7m8 at an MOI of 100K or greater can efficiently deliver transgene into hBMSCs with up to near 100% transduction efficiency for sustained expression over one month. However, donor-to-donor variation exists in transduction efficiency and transgene expression, especially at MOIs less than 100K.

Determining On-Target, Off-Target, and Copy Number Status of Transgenic Events After CRISPR/Cas9 Targeted AAVS1 Safe-Harbor Modification of iPSCs Using Double-Control Quantitative Copy Number PCR.

Double-control quantitative copy number PCR (dc-qcnPCR) is a recently described tool that can be used to quantify donor DNA insertions in genetically modified monoclonal cell lines. In conjunction with an insert-confirmation PCR, the technique can quickly and easily identify clones containing on-target heterozygous or homozygous donor DNA integrations and exclude off-target insertions. The genetic manipulation of immortal cell lines is a versatile tool to elucidate cellular signaling pathways and protein functions. Despite recent advances in the precision of genetic engineering tools such as CRISPR/Cas9, transcription-activator-like effector nucleases (TALENs), and zinc-finger nucleases (ZFNs), it is still essential to verify the accurate insertion of the sequence of interest (donor DNA) into the targeted genomic DNA (gDNA) locus. This precise integration into a genetic safe harbor, and exclusion of the donor DNA from functionally relevant genes, can ensure normal cellular functionality. Current methods to analyze the specificity of donor DNA insertions either are cost-prohibitive or create dependency on manufacturers for assay design and production. The dc-qcnPCR method is a simple, yet powerful, approach that can be prepared and carried out in any laboratory equipped with standard molecular biology supplies. Here we provide step-by-step instructions to prepare and perform the dc-qcnPCR, and its companion insert-confirmation PCR, to determine donor DNA insertion numbers in monoclonal cell lines genetically modified through CRISPR/Cas9. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Genetic modification at AAVS1 safe harbor in induced pluripotent stem cells (IMR90-4) using CRISPR/Cas9: from plasmid design to monoclonal expansion Support Protocol 1: Measurement of Gaussia luciferase activity to verify reporter protein functionality Support Protocol 2: Verification of monoclonal expansion using immunofluorescence. Basic Protocol 2: Insert-confirmation PCR Basic Protocol 3: Design and preparation of double-control quantitative copy number PCR reagents and quantification of donor DNA integrations in genetically modified monoclonal cells.

Near-Infrared-Triggered On-Demand Controlled Release of Adeno-Associated Virus from Alginate Hydrogel Microbeads with Heat Transducer for Gene Therapy.

Gene therapy using adeno-associated virus (AAV) has potential as a radical treatment modality for genetic diseases such as sensorineural deafness. To establish clinical applications, it is necessary to avoid immune response to AAV by controlled release system of AAV. Here, a near-infrared (NIR)-triggered on-demand AAV release system using alginate hydrogel microbeads with a heat transducer is proposed. By using a centrifuge-based microdroplet shooting device, the microbeads encapsulating AAV with Fe3 O4 microparticles (Fe3 O4 -MPs) as a heat transducer are fabricated. Fe3 O4 -MPs generated heat by NIR enhanced the diffusion speed of the AAV, resulting in the AAV being released from the microbeads. By irradiating the microbeads encapsulating fluorescent polystyrene nanoparticles (FP-NPs) (viral model) with NIR, the fluorescence intensity decreased only for FP-NPs with a diameter of 20 nm and not for 100 or 200 nm, confirming that this system can release virus with a diameter of several tens of nanometers. By irradiating NIR to the AAV-encapsulating microbeads with Fe3 O4 -MPs, the AAV is released on demand, and gene transfection to cells by AAV is confirmed without loss of viral activity. The NIR-triggered AAV release system proposed in this study increases the number of alternatives for the method of drug release in gene therapy.

Routes of administration for adeno-associated viruses carrying gene therapies for brain diseases.

Gene therapy is a powerful tool to treat various central nervous system (CNS) diseases ranging from monogenetic diseases to neurodegenerative disorders. Adeno-associated viruses (AAVs) have been widely used as the delivery vehicles for CNS gene therapies due to their safety, CNS tropism, and long-term therapeutic effect. However, several factors, including their ability to cross the blood-brain barrier, the efficiency of transduction, their immunotoxicity, loading capacity, the choice of serotype, and peripheral off-target effects should be carefully considered when designing an optimal AAV delivery strategy for a specific disease. In addition, distinct routes of administration may affect the efficiency and safety of AAV-delivered gene therapies. In this review, we summarize different administration routes of gene therapies delivered by AAVs to the brain in mice and rats. Updated knowledge regarding AAV-delivered gene therapies may facilitate the selection from various administration routes for specific disease models in future research.

High-throughput optical action potential recordings in hiPSC-derived cardiomyocytes with a genetically encoded voltage indicator in the AAVS1 locus.

Cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSCs) represent an excellent in vitro model in cardiovascular research. Changes in their action potential (AP) dynamics convey information that is essential for disease modeling, drug screening and toxicity evaluation. High-throughput optical AP recordings utilizing intramolecular Förster resonance energy transfer (FRET) of the voltage-sensitive fluorescent protein (VSFP) have emerged as a substitute or complement to the resource-intensive patch clamp technique. Here, we functionally validated our recently generated voltage indicator hiPSC lines stably expressing CAG-promoter-driven VSFP in the AAVS1 safe harbor locus. By combining subtype-specific cardiomyocyte differentiation protocols, we established optical AP recordings in ventricular, atrial, and nodal CMs in 2D monolayers using fluorescence microscopy. Moreover, we achieved high-throughput optical AP measurements in single hiPSC-derived CMs in a 3D context. Overall, this system greatly expands the spectrum of possibilities for high-throughput, non-invasive and long-term AP analyses in cardiovascular research and drug discovery.

Efficient Gene Expression in Human Stem Cell Derived-Cortical Organoids Using Adeno Associated Virus.

Cortical organoids are 3D structures derived either from human embryonic stem cells or human induced pluripotent stem cells with their use exploding in recent years due to their ability to better recapitulate the human brain in vivo in respect to organization; differentiation; and polarity. Adeno-associated viruses (AAVs) have emerged in recent years as the vectors of choice for CNS-targeted gene therapy. Here; we compare the use of AAVs as a mode of gene expression in cortical organoids; over traditional methods such as lipofectamine and electroporation and demonstrate its ease-of-use in generating quick disease models through expression of different variants of the central gene-TDP-43-implicated in amyotrophic lateral sclerosis and frontotemporal dementia.

AAV-mediated rescue of Eps8 expression in vivo restores hair-cell function in a mouse model of recessive deafness.

The transduction of acoustic information by hair cells depends upon mechanosensitive stereociliary bundles that project from their apical surface. Mutations or absence of the stereociliary protein EPS8 cause deafness in humans and mice, respectively. Eps8 knockout mice (Eps8 -/- ) have hair cells with immature stereocilia and fail to become sensory receptors. Here, we show that exogenous delivery of Eps8 using Anc80L65 in P1-P2 Eps8 -/- mice in vivo rescued the hair bundle structure of apical-coil hair cells. Rescued hair bundles correctly localize EPS8, WHIRLIN, MYO15, and BAIAP2L2, and generate normal mechanoelectrical transducer currents. Inner hair cells with normal-looking stereocilia re-expressed adult-like basolateral ion channels (BK and KCNQ4) and have normal exocytosis. The number of hair cells undergoing full recovery was not sufficient to rescue hearing in Eps8 -/- mice. Adeno-associated virus (AAV)-transduction of P3 apical-coil and P1-P2 basal-coil hair cells does not rescue hair cells, nor does Anc80L65-Eps8 delivery in adult Eps8 -/- mice. We propose that AAV-induced gene-base therapy is an efficient strategy to recover the complex hair-cell defects in Eps8 -/- mice. However, this therapeutic approach may need to be performed in utero since, at postnatal ages, Eps8 -/- hair cells appear to have matured or accumulated damage beyond the point of repair.

Quantification of transgene expression in GSH AAVS1 with a novel CRISPR/Cas9-based approach reveals high transcriptional variation.

Genomic safe harbors (GSH) are defined as sites in the host genome that allow stable expression of inserted transgenes while having no adverse effects on the host cell, making them ideal for use in basic research and therapeutic applications. Silencing and fluctuations in transgene expression would be highly undesirable effects. We have previously shown that transgene expression in Jurkat T cells is not silenced for up to 160 days after CRISPR-Cas9-mediated insertion of reporter genes into the adeno-associated virus site 1 (AAVS1), a commonly used GSH. Here, we studied fluctuations in transgene expression upon targeted insertion into the GSH AAVS1. We have developed an efficient method to generate and validate highly complex barcoded plasmid libraries to study transgene expression on the single-cell level. Its applicability is demonstrated by inserting the barcoded transgene Cerulean into the AAVS1 locus in Jurkat T cells via the CRISPR-Cas9 technology followed by next-generation sequencing of the transcribed barcodes. We observed large transcriptional variations over two logs for transgene expression in the GSH AAVS1. This barcoded transgene insertion model is a powerful tool to investigate fluctuations in transgene expression at any GSH site.

Targeting the AAVS1 Site by CRISPR/Cas9 with an Inducible Transgene Cassette for the Neuronal Differentiation of Human Pluripotent Stem Cells.

CRISPR/Cas9 system is a powerful genome-editing technology for studying genetics and cell biology. Safe harbor sites are ideal genomic locations for transgene integration with minimal interference in cellular functions. Gene targeting of the AAVS1 locus enables stable transgene expression without phenotypic effects in host cells. Here, we describe the strategy for targeting the AAVS1 site with an inducible Neurogenin-2 (Ngn2) donor template by CRISPR/Cas9 in hiPSCs, which facilitates generation of an inducible cell line that can rapidly and homogenously differentiate into excitatory neurons.

Evaluation of in vitro fibroblast migration by electrospun triple-layered PU-CA/gelatin.PRGF/PU-CA scaffold using an AAVS1 targeted EGFP reporter cell line.

Introduction: Migration of fibroblast cells in wound areas is a critical aspect of the wound healing process. Employment of enhanced green fluorescent protein (EGFP) labeled fibroblast cells facilitates real-time monitoring and functional evaluation of these cells in both in vitro and in vivo settings. Plasma rich in growth factor (PRGF) is a potent accelerator of wound healing; therefore, in this study, a novel method to fabricate an electrospun bioactive scaffold containing PRGF was employed to induce in vitro cell proliferation and migration. Methods: First, the EGFP reporter gene was integrated into the AAVS1 locus of fibroblast cells using CRISPR/Cas9 system. Then, PRGF was obtained from platelet-rich plasma, and a multi-layered scaffold was fabricated using polyurethane-cellulose acetate (PU-CA) fibers as the outer layers and PRGF-containing gelatin fibers were located in the internal layer like a central strip. Scanning electron microscopy (SEM), tensile, water contact angle, and FTIR tests were performed to assess the characteristics of the scaffolds. The EGFP targeted cells were cultured on scaffolds with or without PRGF to investigate their viability, toxicity, and migration pattern in response to the release profile. Results: Fluorescence images showed that the number of migrating cells on scaffold containing PRGF was more significant than PU-CA scaffold up to day 6. Increased expression of SGPL1, DDR2, and VEGF genes was also observed on the scaffold containing PRGF compared to PU-CA using real-time polymerase chain reaction (PCR) analysis with around 3-, 2-, and 2-fold enhancement, respectively. Conclusion: The current scaffold provides the appropriate template for cell attachment and migration. In addition, the present results highlight the potential of reporter gene targeting for the in vitro analysis of biological processes such as migration.