In addressing the limitations of CRISPR-Cas9, including off-target effects and high licensing fees for commercial use, Cas-CLOVER, a dimeric gene editing tool activated by two guide RNAs, was recently developed. This study focused on implementing and evaluating Cas-CLOVER in HEK-293 cells used for recombinant adeno-associated virus (rAAV) production by targeting the signal transducer and activator of transcription 1 (STAT1) locus, which is crucial for cell growth regulation and might influence rAAV production yields. Cas-CLOVER demonstrated impressive efficiency in gene editing, achieving over 90% knockout (KO) success. Thirteen selected HEK-293 STAT1 KO sub-clones were subjected to extensive analytical characterization to assess their genomic stability, crucial for maintaining cell integrity and functionality. Additionally, rAAV9 productivity, Rep protein pattern profile, and potency, among others, were assessed. Clones showed significant variation in capsid and vector genome titers, with capsid titer reductions ranging from 15% to 98% and vector genome titers from 16% to 55%. Interestingly, the Cas-CLOVER-mediated STAT1 KO bulk cell population showed a better ratio of full to empty capsids. Our study also established a comprehensive analytical workflow to detect and evaluate the gene KOs generated by this innovative tool, providing a solid groundwork for future research in precise gene editing technologies.

Adeno-associated viruses (AAVs) are gaining traction as delivery vehicles for gene therapy although the molecular understanding of AAV-transgene release is still limited. Typically, the process of viral uncoating is investigated (in vitro) through thermal stress, revealing capsid disintegration at elevated temperatures. To assess the (in)stability of different empty and filled AAV preparations, we used the light-scattering-based interferometric microscopy technique of mass photometry that, on a single-particle basis, determines the molecular weight of AAVs. By introducing a heat-stable DNA plasmid as an internal standard, we quantitatively probed the impact of heat on AAVs. Generally, empty AAVs exhibited greater heat resistance than genome-filled particles. Our data also indicate that upon DNA release, the capsids do not transform into empty AAVs, but seem to aggregate or disintegrate. Strikingly, some AAVs exhibited an intermediate state with disrupted capsids but preserved bound genome, a feature that experimentally only emerged following incubation with a nuclease. Our data demonstrate that the thermal uncoating process is highly AAV specific (i.e., can be influenced by serotype, genome, host system). We argue that nuclease treatment in combination with MP can be used as an additional analytical tool for assessing structural integrity of recombinant and/or clinical AAV vectors.

Aggregation of the protein α-Synuclein (αSyn) is a hallmark of Parkinson\'s disease (PD), dementia with Lewy bodies (DLB) and multiple systems atrophy, and alleviating the extent of αSyn pathology is an attractive strategy against neurodegeneration. The engineered binding protein β-wrapin AS69 binds monomeric αSyn. AS69 reduces primary and secondary nucleation as well as fibril elongation in vitro. It also mitigates aSyn pathology in a mouse model based on intrastriatal injection of aSyn pre-formed fibrils (PFFs). Since the PFF-based model does not represent all aspects of PD, we tested here whether AS69 can reduce neurodegeneration resulting from αSyn overexpression. Human A53T-αSyn was overexpressed in the mouse Substantia nigra (SN) by using recombinant adeno-associated viral vector (rAAV). AS69 was also expressed by rAAV transduction. Behavioral tests and immunofluorescence staining were used as outcomes. Transduction with rAAV-αSyn resulted in αSyn pathology as reported by phospho-αSyn staining and caused degeneration of dopaminergic neurons in the SN. The co-expression of rAAV-AS69 did not reduce αSyn pathology or the degeneration of dopaminergic neurons. We conclude that αSyn monomer binding by rAAV-AS69 was insufficient to protect from aSyn pathology resulting from αSyn overexpression.

The gene therapy field seeks cost-effective, large-scale production of recombinant adeno-associated virus (rAAV) vectors for high-dosage therapeutic applications. Although strategies like suspension cell culture and transfection optimization have shown moderate success, challenges persist for large-scale applications. To unravel molecular and cellular mechanisms influencing rAAV production, we conducted an SWATH-MS proteomic analysis of HEK293T cells transfected using standard, sub-optimal, and optimal conditions. Gene Ontology and pathway analysis revealed significant protein expression variations, particularly in processes related to cellular homeostasis, metabolic regulation, vesicular transport, ribosomal biogenesis, and cellular proliferation under optimal transfection conditions. This resulted in a 50% increase in rAAV titer compared with the standard protocol. Additionally, we identified modifications in host cell proteins crucial for AAV mRNA stability and gene translation, particularly regarding AAV capsid transcripts under optimal transfection conditions. Our study identified 124 host proteins associated with AAV replication and assembly, each exhibiting distinct expression pattern throughout rAAV production stages in optimal transfection condition. This investigation sheds light on the cellular mechanisms involved in rAAV production in HEK293T cells and proposes promising avenues for further enhancing rAAV titer during production.

Recombinant adeno-associated virus (rAAV) is a major gene delivery vehicle. We have constructed a stable rAAV producer cell line by integrating essential rAAV genome, viral and helper genes into the genome of HEK293 cell under the control of inducible promoters. Upon induction, the cell line produces transducing rAAV. To gain insight into enhancing rAAV productivity and vector quality, we performed a comparative transcriptomic and proteomic analysis of our synthetic cell line GX2 and two wild-type AAV (wtAAV) production systems, one by virus co-infection and the other by multi-plasmid transfection. The three systems had different kinetics in viral component synthesis but generated comparable copies of AAV genomes; however, the capsid titer of GX2 was an order of magnitude lower compared to those two wtAAV systems, indicating that its capsid production may be insufficient. The genome packaging efficiency was also lower in GX2 despite it produced higher levels of Rep52 proteins than either wtAAV systems, suggesting that Rep52 protein expression may not limit genome packaging. In the two wtAAV systems, VP were the most abundant AAV proteins and their levels continued to increase, while GX2 had high level of wasteful cargo gene expression. Furthermore, upregulated inflammation, innate immune responses, and MAPK signaling, as well as downregulated mitochondrial functions, were commonly observed in either rAAV or wtAAV systems. Overall, this comparative multi-omics study provided rich insights into host cell and viral factors that are potential targets for genetic and process intervention to enhance the productivity of synthetic rAAV producer cell lines. KEY POINTS: • wtAAV infection was more efficient in producing full viral particles than the synthetic cell GX2. • Capsid protein synthesis, genome replication, and packaging may limit rAAV production in GX2. • wtAAV infection and rAAV production in GX2 elicited similar host cell responses.

Lafora disease is a rare and fatal form of progressive myoclonic epilepsy typically occurring early in adolescence. The disease results from mutations in the EPM2A gene, encoding laforin, or the EPM2B gene, encoding malin. Laforin and malin work together in a complex to control glycogen synthesis and prevent the toxicity produced by misfolded proteins via the ubiquitin-proteasome system. Disruptions in either protein cause alterations in this complex, leading to the formation of Lafora bodies containing abnormal, insoluble, and hyperphosphorylated forms of glycogen. We used the Epm2a-/- knockout mouse model of Lafora disease to apply gene therapy by administering intracerebroventricular injections of a recombinant adeno-associated virus carrying the human EPM2A gene. We evaluated the effects of this treatment through neuropathological studies, behavioral tests, video-electroencephalography, electrophysiological recordings, and proteomic/phosphoproteomic analysis. Gene therapy ameliorated neurological and histopathological alterations, reduced epileptic activity and neuronal hyperexcitability, and decreased the formation of Lafora bodies. Moreover, differential quantitative proteomics and phosphoproteomics revealed beneficial changes in various molecular pathways altered in Lafora disease. Our results represent proof of principle for gene therapy with the coding region of the human EPM2A gene as a treatment for EPM2A-related Lafora disease.

Recombinant adeno-associated virus (rAAV) has emerged as a prominent vector for in vivo gene therapy, owing to its distinct advantages. Accurate determination of the rAAV genome titer is crucial for ensuring the safe and effective administration of clinical doses. The evolution of the rAAV genome titer assay from quantitative PCR (qPCR) to digital PCR (dPCR) has enhanced accuracy and precision, yet practical challenges persist. This study systematically investigated the impact of various operational factors on genome titration in a single-factor manner, aiming to address potential sources of variability in the quantitative determination process. Our findings revealed that a pretreatment procedure without genome extraction exhibits superior precision compared with titration with genome extraction. Additionally, notable variations in titration results across different brands of dPCR instruments were documented, with relative standard deviation (RSD) reaching 23.47% for AAV5 and 11.57% for AAV8. Notably, optimal operations about DNase I digestion were identified; we thought treatment time exceeding 30 min was necessary, and there was no need for thermal inactivation after digestion. And we highlighted that thermal capsid disruption before serial dilution substantially affected AAV genome titers, causing a greater than ten-fold decrease. Conversely, this study found that additive components of dilution buffer are not significant contributors to titration variations. Furthermore, we found that repeated freeze-thaw cycles significantly compromised AAV genome titers. In conclusion, a comprehensive dPCR titration protocol, incorporating insights from these impact factors, was proposed and successfully tested across multiple serotypes of AAV. The results demonstrate acceptable variations, with the RSD consistently below 5.00% for all tested AAV samples. This study provides valuable insights to reduce variability and improve the reproducibility of AAV genome titration using dPCR.

The production of recombinant adeno-associated virus (rAAV) for gene therapy applications relies on the use of various host cell lines, with suspension-grown HEK293 cells being the preferred expression system due to their satisfactory rAAV yields in transient transfections. As the field of gene therapy continues to expand, there is a growing demand for efficient rAAV production, which has prompted efforts to optimize HEK293 cell line productivity through engineering. In contrast to other cell lines like CHO cells, the transcriptome of HEK293 cells during rAAV production has remained largely unexplored in terms of identifying molecular components that can enhance yields. In our previous research, we analyzed global regulatory pathways and mRNA expression patterns associated with increased rAAV production in HEK293 cells. Our data revealed substantial variations in the expression patterns between cell lines with low (LP) and high-production (HP) rates. Moving to a deeper layer for a more detailed analysis of inflammation-related transcriptome data, we detected an increased expression of interferon-related genes in low-producing cell lines. Following upon these results, we investigated the use of Ruxolitinib, an interferon pathway inhibitor, during the transient production of rAAV in HEK293 cells as potential media additive to boost rAAV titers. Indeed, we find a two-fold increase in rAAV titers compared to the control when the interferon pathways were inhibited. In essence, this work offers a rational design approach for optimization of HEK293 cell line productivity and potential engineering targets, ultimately paving the way for more cost-efficient and readily available gene therapies for patients.

Recombinant adeno-associated viruses (rAAVs) stand at the forefront of gene therapy applications, holding immense significance for their safe and efficient gene delivery capabilities. The constantly increasing and unmet demand for rAAVs underscores the need for a more comprehensive understanding of AAV biology and its impact on rAAV production. In this literature review, we delved into AAV biology and rAAV manufacturing bioprocesses, unravelling the functions and essentiality of proteins involved in rAAV production. We discuss the interconnections between these proteins and how they affect the choice of rAAV production platform. By addressing existing inconsistencies, literature gaps and limitations, this review aims to define a minimal set of genes that are essential for rAAV production, providing the potential to advance rAAV biomanufacturing, with a focus on minimizing the genetic load within rAAV-producing cells.

The development of bone-targeting drug delivery systems holds immense promise for improving the treatment of skeletal diseases. By precisely delivering therapeutic agents to the affected areas of bone, these strategies can enhance drug efficacy, minimize off-target effects, and promote patient adherence, ultimately leading to improved treatment outcomes and an enhanced quality of life for patients. This review aims to provide an overview of the current state of affinity-based bone-targeting agents and recent breakthroughs in innovative bone-targeting adeno-associated virus (AAV) strategies to treat skeletal diseases in mice. In particular, this review will delve into advanced AAV engineering, including AAV serotype selection for bone targeting and capsid modifications for bone-specific tropism. Additionally, we will highlight recent advancements in AAV-mediated gene therapy for skeletal diseases and discuss challenges and future directions of this promising therapeutic approach.