Recombinant Adeno-associated Virus Vectors Research Articles

Effects of rAAV-Mediated Overexpression of sox9 and TGF-ß via Alginate Hydrogel-Guided Vector Delivery on the Chondroreparative Activities of Human Bone Marrow-Derived Mesenchymal Stromal Cells

Recombinant adeno-associated virus (rAAV) vectors have a strong potential to promote the healing of traumatic cartilage defects and osteoarthritic lesions upon delivery and overexpression of therapeutic genes from suitable biomaterials that support a controlled release of the candidate constructs. The goal of the present work is to examine whether the administration of chondrogenic rAAV sox9 and rAAV TGF-ß gene vehicles via alginate hydrogel-guided vector delivery stimulates the biological and chondroreparative activities of human bone marrow-derived mesenchymal stromal cells (hMSCs) as a source of improved reparative cells for future implantation in sites of cartilage damage. The delivery of rAAV using an alginate (AlgPH155) hydrogel system is successfully achieved in hMSCs over time (21 days), leading to the effective overexpression of sox9 and TGF-ß that significantly increases the proliferation and chondrogenic differentiation activities of the cells relative to control (marker lacZ) gene transfer while advantageously preventing premature hypertrophy, osteogenesis, and mineralization. This study reveals the potential of alginate hydrogel-based systems to control the delivery of rAAV (sox9 and TGF-ß) gene vectors to adeptly trigger the chondroreparative activities of hMSCs for future applications that aim at improving cartilage repair.

A mosaic adeno-associated virus vector as a versatile tool that exhibits high levels of transgene expression and neuron specificity in primate brain

Recent emphasis has been placed on gene transduction mediated through recombinant adeno-associated virus (AAV) vector to manipulate activity of neurons and their circuitry in the primate brain. In the present study, we created a novel vector of which capsid was composed of capsid proteins derived from both of the AAV serotypes 1 and 2 (AAV1 and AAV2). Following the injection into the frontal cortex of macaque monkeys, this mosaic vector, termed AAV2.1 vector, was found to exhibit the excellence in transgene expression (for AAV1 vector) and neuron specificity (for AAV2 vector) simultaneously. To explore its applicability to chemogenetic manipulation and in vivo calcium imaging, the AAV2.1 vector expressing excitatory DREADDs or GCaMP was injected into the striatum or the visual cortex of macaque monkeys, respectively. Our results have defined that such vectors secure intense and stable expression of the target proteins and yield conspicuous modulation and imaging of neuronal activity.

Abstract P3059: Recombinant Adeno-associated Viral Vector 2/8 Model Of Interleukin 6 Mediated Chronic Inflammation

Background: Interleukin 6 (IL6) is a pleiotropic cytokine mostly known as a proinflammatory cytokine. There is a need for a reliable in vivo model to study IL6 mediated chronic inflammation. Hypothesis: The aim of our study was to create a mouse model mimicking the chronic inflammatory state as observed in cancer and inflammatory diseases. We opted for a recombinant adeno-associated viral vector (rAAV) to mimic the acute initiation of chronic inflammation later in life. Methods: We opted for a rAAV2/8 subtype, human EF1a short (EFS) promoter and firefly luciferase led by an internal ribosome entry site (IL6-IRES-fLuc) DNA cassette to create a stable low-grade expression of IL6 in liver and serum, with minimal expression in other organs, particularly the heart. rAAV2/8_EFS-IL6-IRES-fLuc and the control vector rAAV2/8_EFS-MCS-IRES-fLuc were generated and different dosages were tested. Results: All mice treated with high dose AAV2/8-IL6 vector (8.8* 10 10 genome copies (GC)/animal) showed severe weight loss and hepatosplenomegaly, starting between week 4-6 post injection. Histological examination of the liver showed features of chronic hepatitis. Infiltrating cells were found in both the liver and the heart. In the spleen, the histological structure of red and white pulp was disturbed and more giant cells could be detected. Cardiac function remained normal at 6 weeks post injection. Assuming that the previous results were due to an overexpression of IL6 due to too high amounts of rAAV2/8_EFS-IL6-IRES-fLuc, we subsequently challenged mice with a rAAV2/8_EFS-IL6-IRES-fLuc dilution series to test which vector dose could be used for reliable long-term low grade IL6 overexpression. After extensive testing, the ideal dose for a 12 weeks IL6 overexpression model was between 2.2 and 2.93* 10 10 GC. Using this dose range, we saw stable overexpression of IL6 without weight loss. Hepatosplenomegaly, disturbed anatomy of the spleen and infiltrating cells in the liver were still present yet less pronounced. Conclusion: We successfully generated and characterized a mouse model of IL6 overexpression to mimic chronic IL6 mediated inflammation. To the best of our knowledge this is the first model employing IL6 overexpression using a rAAV viral vector.

Combined clarification and affinity capture using magnetic resin enables efficient separation of rAAV5 from cell lysate

Recombinant adeno-associated virus (rAAV) vectors have displayed enormous potential as a platform for delivery of gene therapies. Purification of rAAV at industrial scale involves a series of elaborate, material, and time-consuming midstream steps, such as clarification by depth filtration and concentration/buffer exchange by tangential flow filtration. In this study, we developed a filter-less flow capture method for purification of rAAV serotype 5, using a high-gradient magnetic separator and magnetic Mag Sepharose beads coupled to an AVB affinity ligand. In under 2 h, we captured and eluted rAAV5 directly from ∼5L of cell lysate with a recovery yield of 63% (±5%, n= 3). Compared to cell lysate, the eluate showed a 3-log reduction of host cell DNA and host cell proteins. The process developed eliminates the need for filtration and column chromatography in the early steps of industrial rAAV purification. This will be of high value for industrial-scale manufacturing of rAAVs by reducing time and material in the purification process, without compromising product recovery and purity.

Design space determination to optimize DNA complexation and full capsid formation in transient rAAV manufacturing.

Recombinant adeno-associated virus (rAAV) vectors are a promising platform for in vivo gene therapies. However, cost-effective, well-characterized processes necessary to manufacture rAAV therapeutics are challenging to develop without an understanding of how process parameters (PPs) affect rAAV product quality attributes (PQAs). In this work, a central composite orthogonal experimental design was employed to examine the influence of four PPs for transient transfection complex formation (polyethylenimine:DNA [PEI:DNA] ratio, total DNA/cell, cocktail volume, and incubation time) on three rAAV PQAs related to capsid content (vector genome titer, vector genome:capsid particle ratio, and two-dimensional vector genome titer ratio). A regression model was established for each PQA using partial least squares, and a design space (DS) was defined in which Monte Carlo simulations predicted < 1% probability of failure (POF) to meet predetermined PQA specifications. Of the three PQAs, viral genome titer was most strongly correlated with changes in complexation PPs. The DS and acceptable PP ranges were largest when incubation time and cocktail volume were kept at mid-high setpoints, and PEI:DNA ratio and total DNA/cell were at low-mid setpoints. Verification experiments confirmed model predictive capability, and this work establishes a framework for studying other rAAV PPs and their relationship to PQAs.

Facing potential chemistry, manufacturing, &amp; control (CMC) development challenges with recombinant adeno-associated viral vectors: available regulatory guidance &amp; recommendations

Facing potential chemistry, manufacturing, &amp; control (CMC) development challenges with recombinant adeno-associated viral vectors: available regulatory guidance &amp; recommendations

VWA3A-derived ependyma promoter drives increased therapeutic protein secretion into the CSF

Recombinant adeno-associated viral vectors (rAAVs) are a promising strategy to treat neurodegenerative diseases because of their ability to infect non-dividing cells and confer long-term transgene expression. Despite an ever-growing library of capsid variants, widespread delivery of AAVs in the adult central nervous system remains a challenge. We have previously demonstrated successful distribution of secreted proteins by infection of the ependyma, a layer of post-mitotic epithelial cells lining the ventricles of the brain and central column of the spinal cord, and subsequent protein delivery via the cerebrospinal fluid (CSF). Here we define a functional ependyma promoter to enhance expression from this cell type.Using RNA sequencing on human autopsy samples, we identified disease- and age-independent ependyma gene signatures. Associated promoters were cloned and screened aslibraries in mouse and rhesus macaque to reveal cross-species function of a human DNA-derived von Willebrand factordomain containing 3A (VWA3A) promoter. When tested in mice, our VWA3A promoter drove strong, ependyma-localized expression of eGFP and increased secreted ApoE protein levels in the CSF by 2-12× over the ubiquitous iCAG promoter.

Liver directed adeno-associated viral vectors to treat metabolic disease.

The liver is the metabolic center of the body and an ideal target for gene therapy of inherited metabolic disorders (IMDs). Adeno-associated viral (AAV) vectors can deliver transgenes to the liver with high efficiency and specificity and a favorable safety profile. Recombinant AAV vectors contain only the transgene cassette, and their payload is converted to non-integrating circular double-stranded DNA episomes, which can provide stable expression from months to years. Insights from cellular studies and preclinical animal models have provided valuable information about AAV capsid serotypes with a high liver tropism. These vectors have been applied successfully in the clinic, particularly in trials for hemophilia, resulting in the first approved liver-directed gene therapy. Lessons from ongoing clinical trials have identified key factors affecting efficacy and safety that were not readily apparent in animal models. Circumventing pre-existing neutralizing antibodies to the AAV capsid, and mitigating adaptive immune responses to transduced cells are critical to achieving therapeutic benefit. Combining the high efficiency of AAV delivery with genome editing is a promising path to achieve more precise control of gene expression. The primary safety concern for liver gene therapy with AAV continues to be the small risk of tumorigenesis from rare vector integrations. Hepatotoxicity is a key consideration in the safety of neuromuscular gene therapies which are applied at substantially higher doses. The current knowledge base and toolkit for AAV is well developed, and poised to correct some of the most severe IMDs with liver-directed gene therapy.

CircSnd1 promotes atherosclerosis progression through the miR-485-3p/Olr1 signaling pathway

BackgroundCircular RNAs (circRNAs) participate in the development of atherosclerotic cardiovascular disease. Identifying and verifying the key competing endogenous RNA (ceRNA) network related to atherosclerosis (AS) is significant for understanding the development of AS. The aim of this study was to investigate the circRNA-miRNA‒mRNA network, identify a key circRNA and explore its role in the development of atherosclerosis. MethodsDifferentially expressed mRNAs (DEMs) and circRNAs (DECs) in the AS model were obtained from datasets in the Gene Expression Omnibus (GEO) database. R software and Cytoscape software were used to construct and visualize the ceRNA network. The dual-luciferase reporter experiment and the RNA pull-down experiment were used to verify the selected ceRNA axis. siRNA targeting circRNA, miRNA mimic, miRNA inhibitor, or gene overexpression plasmid was used for in vitro functional studies. ELISA and western blotting were used to detect inflammation and lipid transport-related proteins. Furthermore, an AS mouse model was established and treated with recombinant adeno-associated viral vectors to further verify the influence of the selected ceRNA axis on the occurrence and/or development of AS. ResultsA total of 497 DEMs were enriched in 25 pathways, based on which the circ_0082139 (circSnd1)/miR-485-3p/Olr1 axis was selected. In vitro, the interaction among the three molecules of this axis was validated and it was found to affect inflammation and lipid transport, which were characterized by the significant change of inflammatory factors (Il-6, Il-8, Tnf-α, Mcp-1, Vcam-1, and Icam-1), and lipid transport-related genes, including Abca1, Abcg1, Ldlr, Hdlbp, Lp-pla2, and Srebp-1c. Through animal experiments, we further verified that the circSnd1/miR-485-3p/Olr1 axis regulated these molecules and participated in the formation and/or development of AS in vivo. ConclusionsThe circSnd1/miR-485-3p/Olr1 axis participates in the formation and development of atherosclerosis by regulating inflammation and lipid transport.

Improving retinal vascular endothelial cell tropism through rational rAAV capsid design.

Vascular endothelial cells (VEC) are essential for retinal homeostasis and their dysfunction underlies pathogenesis in diabetic retinopathy (DR) and exudative age-related macular degeneration (AMD). Studies have shown that recombinant adeno-associated virus (rAAV) vectors are effective at delivering new genetic material to neural and glial cells within the retina, but targeting VECs remains challenging. To overcome this limitation, herein we developed rAAV capsid mutant vectors with improved tropism towards retinal VEC. rAAV2/2, 2/2[QuadYF-TV], and rAAV2/9 serotype vectors (n = 9, capsid mutants per serotype) expressing GFP were generated by inserting heptameric peptides (7AA) designed to increase endothelial targeting at positions 588 (2/2 and 2/2[QuadYF-TV] or 589 (2/9) of the virus protein (VP 1-3). The packaging and transduction efficiency of the vectors were assessed in HEK293T and bovine VECs using Fluorescence microscopy and flow cytometry, leading to the identification of one mutant, termed EC5, that showed improved endothelial tropism when inserted into all three capsid serotypes. Intra-ocular and intravenous administration of EC5 mutants in C57Bl/6j mice demonstrated moderately improved transduction of the retinal vasculature, particularly surrounding the optic nerve head, and evidence of sinusoidal endothelial cell transduction in the liver. Most notably, intravenous administration of the rAAV2/2[QuadYF-TV] EC5 mutant led to a dramatic and unexpected increase in cardiac muscle transduction.

Abstract 513: Localised Anticoagulation Via Gene Therapy: Towards Prevention Of Cardioembolic Stroke

Introduction: The coagulation cascade thrives as the haematological gatekeeper ensuring equilibrium between blood flow and thrombosis. Under pathological conditions, the positive-feedback loops compound upon one another, disrupting this equilibrium and increasing the risk of thrombosis and embolic events. Cardiac thromboembolism persists as the culprit behind most ischemic strokes; a majority arising from the left atrial appendage. In order to combat this disease and overcome the limitations of systemic oral anticoagulants, we developed a novel anticoagulant viral vector to focally target cardiac regions of increased thrombotic potential. Hypothesis: Our novel recombinant lentiviral and adeno-associated viral vectors expressing thrombomodulin will induce a significant cell autonomous anticoagulant effect when expressed by endothelial cells of the left atrial appendage. Methods: The anticoagulant phenotype of these vectors was functionally assessed through in vitro and ex vivo platforms. For in vitro testing, a novel, cell-based version of the calibrated automated thrombogram assay, coupled with the overall haemostatic potential assay were utilised to determine thrombokinetics in cell culture models. For ex vivo testing, freshly isolated and characterized porcine left atrial appendage endothelial cells were used. These cells were used to endothelialise microfluidic devices, which were stimulated with TNF-α prior to perfusion with human whole blood. Changes in fibrin, platelets and neutrophils were investigated in the presence and absence of our novel anticoagulant viral vectors. Results: Experimental findings from our in vitro and ex vivo work showed a significant decrease in the endogenous thrombin potential of vector-transduced cells, coupled with a significant decrease in the velocity of thrombogenesis. Furthermore, transduced cells had a marked decrease in the peak rate of thrombin generation and a significant increase in the time taken to achieve peak thrombin generation. Conclusion: These findings show the impact of our anticoagulant vectors through inhibiting thrombosis and guide the development of focal gene therapy strategies to target thrombosis at its point of origin in the prevention of cardioembolic-stroke.

Investigating HIV-1 TAT protein role in Atherogenesis

Abstract With the recent development of antiretroviral therapy, HIV patients can live a long and normal lifespan. Recent evidence demonstrates an increased incidence of developing cardiovascular diseases (CVD) specifically atherosclerosis in aged HIV-infected patients. The adaptive and innate immune responses play a complex specific role in atherosclerotic plaque burden. However, specific mechanisms of the implication of the immune system in HIV-accelerated atherosclerosis are unclear. The protein Trans-Activator of Transcription–TAT is strongly associated with HIV infection as it enhances the efficiency of viral transcription. To understand the contributions of TAT protein in HIV-associated atherogenesis, a doxycycline-inducible astrocyte-specific HIV-1 TAT transgenic mouse was used. An injection of recombinant adeno-associated virus vector (AAV) encoding a gain-of-function mutant PCSK9 into mice and feeding with high fat with added cholesterol (DDC) and doxycycline (Dox) was used to promote atherosclerosis and TAT expression. After 16 wks of feeding, various tissues were collected and used for flow cytometry analysis (FACS) for immune profiling. Our preliminary data suggest that TAT is involved in the regulation of peripheral blood neutrophil numbers and therefore can serve as a regulator of inflammation in HIV-associated atherosclerosis. Future analysis will provide insight into potential mechanisms of HIV TAT-dependent involvement in atherosclerosis development and progression. This work was supported by project –initiation funds from the Center for Integrative Neuroscience and Inflammatory Diseases at EVMS and NIH NHLBI R01HL142129-04S1 grant.

The AAV capsid can influence the epigenetic marking of rAAV delivered episomal genomes in a species dependent manner

Recombinant adeno-associated viral vectors (rAAVs) are among the most commonly used vehicles for in vivo based gene therapies. However, it is hard to predict which AAV capsid will provide the most robust expression in human subjects due to the observed discordance in vector-mediated transduction between species. In our study, we use a primate specific capsid, AAV-LK03, to demonstrate that the limitation of this capsid towards transduction of mouse cells is unrelated to cell entry and nuclear transport but rather due to depleted histone H3 chemical modifications related to active transcription, namely H3K4me3 and H3K27ac, on the vector DNA itself. A single-amino acid insertion into the AAV-LK03 capsid enables efficient transduction and the accumulation of active-related epigenetic marks on the vector chromatin in mouse without compromising transduction efficiency in human cells. Our study suggests that the capsid protein itself is involved in driving the epigenetic status of the vector genome, most likely during the process of uncoating. Programming viral chromatin states by capsid design may enable facile DNA transduction between vector and host species and ultimately lead to rational selection of AAV capsids for use in humans.

Analytical ultracentrifugation sedimentation velocity for the characterization of recombinant adeno-associated virus vectors sub-populations

Recombinant adeno-associated virus virus-derived vectors (rAAVs) are among the most used viral delivery system for in vivo gene therapies with a good safety profile. However, rAAV production methods often lead to a heterogeneous vector population, in particular with the presence of undesired empty particles. Analytical ultracentrifugation sedimentation velocity (AUC-SV) is considered as the gold analytical technique allowing the measurement of relative amounts of each vector subpopulation and components like particle aggregates, based on their sedimentation coefficients. This letter presents the principle and practice of AUC experiments for rAAVs characterization. We discuss our results in the framework of previously published works. In addition to classical detection at 260nm, using interference optics in the ultracentrifuge can provide an independent estimate of weight percentages of the different populations of capsids, and of the genome size incorporated in rAAV particles.

Review of cardiac safety in onasemnogene abeparvovec gene replacement therapy: translation from preclinical to clinical findings

Human gene replacement therapies such as onasemnogene abeparvovec (OA) use recombinant adeno-associated virus (rAAV) vectors to treat monogenic disorders. The heart and liver are known target organs of toxicity in animals; with cardiac and hepatic monitoring recommended in humans after OA dosing. This manuscript provides a comprehensive description of cardiac data from preclinical studies and clinical sources including clinical trials, managed access programs and the post-marketing setting following intravenous OA administration through 23 May 2022. Single dose mouse GLP-Toxicology studies revealed dose-dependent cardiac findings including thrombi, myocardial inflammation and degeneration/regeneration, which were associated with early mortality (4-7 weeks) in the high dose groups. No such findings were documented in non-human primates (NHP) after 6 weeks or 6 months post-dose. No electrocardiogram or echocardiogram abnormalities were noted in NHP or humans. After OA dosing, some patients developed isolated elevations in troponin without associated signs/symptoms; the reported cardiac adverse events in patients were considered of secondary etiology (e.g. respiratory dysfunction or sepsis leading to cardiac events). Clinical data indicate cardiac toxicity observed in mice does not translate to humans. Cardiac abnormalities have been associated with SMA. Healthcare professionals should use medical judgment when evaluating the etiology and assessment of cardiac events post OA dosing so as to consider all possibilities and manage the patient accordingly.

Eladocagene Exuparvovec Gene Therapy Improves Motor Development in Patients With Aromatic L-Amino Acid Decarboxylase Deficiency (S34.004)

<h3>Objective:</h3> Investigate the efficacy and safety of eladocagene exuparvovec in patients with aromatic l-amino acid decarboxylase deficiency (AADCd). <h3>Background:</h3> AADCd results from mutations in the dopa decarboxylase gene leading to reduced AADC enzyme activity; it is characterized by motor impairments and inability to attain developmental milestones. Eladocagene exuparvovec is a recombinant adeno-associated viral vector serotype 2 carrying the coding sequence for human AADC. <h3>Design/Methods:</h3> Eladocagene exuparvovec was infused bilaterally in the putamina of 30 children with AADC deficiency in 3 studies (AADC-CU/1601 [n=8], AADC-010 [n=10], and AADC-011 [n=12] in patients aged 18–102 months. Data were extracted on January 4, 2022. Patients receiving a total of 1.8 × 10¹¹ vg (n=21) or 2.4 × 10¹¹ vg (n=9; AADC-011) were followed for up to 120 months and assessed for motor milestone attainment using the Peabody Developmental Motor Scale, 2nd edition (PDMS-2). Specific motor skill items of the PDMS-2 were used to assess motor milestones including head control (partial or full), sitting (supported or independently), standing (with/away from support; up from cross-legged position), and walking (with/without assistance; 10 feet; taped line) Motor milestones and development were measured every 3 months for 1 year following gene therapy, then every 6–12 months for ≤120 months. <h3>Results:</h3> At baseline, no patients mastered head control or more advanced milestones. At year 1 of follow-up, patients were gaining the following skills (n): partial head control (26); full head control (15), sitting unassisted (7), supported standing (2). Progression of development was noted at years 5 and 10. By year 5, more advanced milestones were achieved (n): full head control (24), sitting unassisted (21) assisted walking (5), walking 10 feet (3), or walking up stairs (3). These abilities were maintained for as long as 10 years <h3>Conclusions:</h3> These data indicate eladocagene exuparvovec can provide a durable, positive impact on motor development in patients with AADCd. <b>Disclosure:</b> Mrs. Russell has received personal compensation for serving as an employee of PTC Therapeutics. Mrs. Russell has stock in PTC Therapeutics. Prof. ROUBERTIE has received personal compensation in the range of $500-$4,999 for serving as a Consultant for PTC. Prof. ROUBERTIE has received personal compensation in the range of $0-$499 for serving on a Scientific Advisory or Data Safety Monitoring board for PTC. Yin-Hsiu Chien has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Sanofi Genzyme. Yin-Hsiu Chien has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Biogen. Yin-Hsiu Chien has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Novartis. Yin-Hsiu Chien has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Amicus. Yin-Hsiu Chien has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Sanofi. Yin-Hsiu Chien has received personal compensation in the range of $500-$4,999 for serving on a Speakers Bureau for Sanofi Genzyme. Yin-Hsiu Chien has received personal compensation in the range of $500-$4,999 for serving on a Speakers Bureau for Avexis/Norvatis. Yin-Hsiu Chien has received personal compensation in the range of $500-$4,999 for serving on a Speakers Bureau for Biogen. The institution of Yin-Hsiu Chien has received research support from Sanofi Genzyme. Ni-Chung Lee has nothing to disclose. Dr. Sierra has received personal compensation for serving as an employee of PTC Therapeutics Inc. Dr. Sierra has stock in PTC Therapeutics Inc. Dr. Turna has received personal compensation for serving as an employee of PTC Therapeutics. Dr. Turna has stock in PTC Therapeutics. Dr. Wang has received personal compensation for serving as an employee of PTC Therapeutics. Dr. Wang has received stock or an ownership interest from PTC Therapeutics, Inc.. The institution of Chun-Hwei Tai has received research support from National Taiwan University Hospital. Paul Wuh-Liang Hwu has received personal compensation in the range of $500-$4,999 for serving as a Consultant for PTC Therapeutics Inc.. Paul Wuh-Liang Hwu has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for PTC therapeutics. The institution of Paul Wuh-Liang Hwu has received research support from PTC therapeutics.

RAAV2-Mediated Restoration of GALC in Neural Stem Cells from Krabbe Patient-Derived iPSCs.

Krabbe disease is a rare neurodegenerative fatal disease. It is caused by deficiency of the lysosomal enzyme galactocerebrosidase (GALC), which results in progressive accumulation of galactolipid substrates in myelin-forming cells. However, there is still a lack of appropriate neural models and effective approaches for Krabbe disease. We generated induced pluripotent stem cells (iPSCs) from a Krabbe patient previously. Here, Krabbe patient-derived neural stem cells (K-NSCs) were induced from these iPSCs. By using nine kinds of recombinant adeno-associated virus (rAAV) vectors to infect K-NSCs, we found that the rAAV2 vector has high transduction efficiency for K-NSCs. Most importantly, rAAV2-GALC rescued GALC enzymatic activity in K-NSCs. Our findings not only establish a novel patient NSC model for Krabbe disease, but also firstly indicate the potential of rAAV2-mediated gene therapy for this devastating disease.

AAV analysis by sedimentation velocity analytical ultracentrifugation: beyond empty and full capsids

The recent surge of therapeutic interest in recombinant adeno-associated viral (AAV) vectors for targeted DNA delivery has brought analytical ultracentrifugation (AUC) into the spotlight. A major concern during formulation of AAV therapeutics is purity of the active species (DNA-containing capsid, or "filled capsids"). Insertion of DNA into AAV is not a highly efficient process; thus, a significant amount of empty and partial/intermediate AAV molecules may exist. Recent guidance from the FDA includes limiting the presence of empty AAV capsids and other impurities to reduce immunotoxicity. While chromatographic techniques (SEC, SEC-MALS, AEX) are often used for empty and full capsid quantitation due to the ease of accessibility and familiarity among most biochemists, the resolution and sensitivity attained by sedimentation velocity (SV-AUC) in the formulation buffer and purification buffers is unmatched. Approaches for using SV-AUC to determine the empty-to-full capsid ratio have already been discussed by others; however, in this report, we focus on the importance of characterizing other impurities, such as free DNA, partially filled capsids, and aggregates that are recognized as species of concern for immunotoxicity. We also demonstrate the usefulness of applying multiple analyses (e.g., c(s), g(s*), WDA) in confirming the presence of and determining the hydrodynamic parameters of these various species.

Expression of the primate-specific LINC00473 RNA in mouse neurons promotes excitability and CREB-regulated transcription

The LINC00473 (Lnc473) gene has previously been shown to be associated with cancer and psychiatric disorders. Its expression is elevated in several types of tumors and decreased in the brains of patients diagnosed with schizophrenia or major depression. In neurons, Lnc473 transcription is strongly responsive to synaptic activity, suggesting a role in adaptive, plasticity-related mechanisms. However, the function of Lnc473 is largely unknown. Here, using a recombinant adeno-associated viral vector, we introduced a primate-specific human Lnc473 RNA into mouse primary neurons. We show that this resulted in a transcriptomic shift comprising downregulation of epilepsy-associated genes and a rise in cAMP response element-binding protein (CREB) activity, which was driven by augmented CREB-regulated transcription coactivator 1 nuclear localization. Moreover, we demonstrate that ectopic Lnc473 expression increased neuronal excitability as well as network excitability. These findings suggest that primates may possess a lineage-specific activity-dependent modulator of CREB-regulated neuronal excitability.

Optimization of Electroporation and Adeno-Associated Virus-Mediated Generation of 2.7 kb Knock-In Livestock Blastocysts

The production of genome-edited livestock has traditionally relied on either somatic cell nuclear transfer cloning of an edited cell or microinjection of gene-editing reagents into developing embryos. Electroporation of gene-editing reagents into zygotes offers an alternative that is both high throughput and easy to use. Transfer of large (>1 kb) homology-directed repair (HDR) donor nucleic acid templates into mammalian zygotes is hindered by the thick extracellular zona pellucida (ZP) glycoprotein layer. A potentially scalable approach to deliver HDR templates of up to 4.9 kb into zygotes is to use recombinant adeno-associated virus (rAAV) vectors. In this study, electroporation conditions were optimized to efficiently deliver CRISPR-associated protein 9 (Cas9):single guide RNA (sgRNA) ribonucleoprotein (RNP) complexes to bovine and ovine zygotes, while retaining embryo viability. rAAV vectors were then used to transduce a 3.9-kb HDR template in combination with electroporation of Cas9:sgRNA RNP editing reagents 6 h postinsemination to generate bovine blastocysts with a targeted 2.7 kb knock-in (KI) at the H11 safe harbor locus. With this approach, there was no need to remove or weaken the ZP of the blastocysts that developed, and a KI rate of up to ∼38% was observed. However, to circumvent transduction of the cumulus cells, the oocytes were denuded before transduction that decreased the blastocyst development rate. In addition, genetic mosaicism was observed in the blastocysts. These results highlight that further optimization of editing approaches will be required to achieve nonmosaic embryos with a targeted KI at scale for livestock applications.