Recombinant Virus Research Articles

MiRNA-Mediated Mechanisms in the Generation of Effective and Safe Oncolytic Viruses.

MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression by inhibiting the translation of target transcripts. The expression profiles of miRNAs vary in different tissues and change with the development of diseases, including cancer. This feature has begun to be used for the modification of oncolytic viruses (OVs) in order to increase their selectivity and efficacy. OVs represent a relatively new class of anticancer drugs; they are designed to replicate in cancer tumors and destroy them. These can be natural viruses that can replicate within cancer tumor cells, or recombinant viruses created in laboratories. There are some concerns regarding OVs' toxicity, due to their ability to partially replicate in healthy tissues. In addition, lytic and immunological responses upon OV therapy are not always sufficient, so various OV editing methods are used. This review discusses the latest results of preclinical and clinical studies of OVs, modifications of which are associated with the miRNA-mediated mechanism of gene silencing.

Maca (Lepidium meyenii Walp.) inhibits HIV-1 infection through the activity of thiadiazole alkaloids in viral integration

Ethnopharmacology relevanceLepidium meyenii Walp. (maca) has been traditionally used for centuries in the Central Andes region both as food and as medicine. In the last decades, its fertility enhancer properties have gained importance, with the majority of the scientific literature related to this topic. However, other traditional uses are less known as metabolic or infectious diseases. Aim of the studyThe main purpose of this study is to investigate the anti-infectious activity of L. meyenii, specifically in HIV-1 infection. There are previous reports of the transcriptional related activity of L. meyenii extracts in human T lymphocytes via transcription factors as NF-κB. Since T lymphocytes are the main target of HIV-1 infection and NF-κB is strongly involved in HIV-1 transcription, L. meyenii could display antiviral activity. Material and methodsChromatography and spectroscopy techniques were used to isolate and identify the compounds in the active extracts. An antiviral assay system based on recombinant viruses was used to evaluate the anti-HIV activity. Cell toxicity was tested for all the extracts and compounds. Viral entry was studied using VSV-HIV chimera viruses and reverse transcription and viral integration were studied by qPCR of viral DNA in infected cells. Finally, viral transcription was studied in primary lymphocytes transfected with HIV-1 or NF-κB luciferase reporter plasmids. Resultsn-Hexane extracts of purple maca displayed anti-HIV activity in an in vitro assay. A bioassay-guided fractionation led to the identification of three thiadiazole alkaloids with antiviral activity. All the compounds were able to inhibit HIV infection of MT-2 cell lines and primary lymphocytes (PBMCs) with IC50 values in the low micromolar range. The mechanism of action differs between the three compounds: one of them showed activity on viral entry, and all the three compounds inhibited viral integration at low concentrations. Remarkably, none of the compounds inhibited reverse transcription or viral transcription. Conclusionsn-Hexane extracts of the purple ecotype of L. meyenii inhibit HIV-1 infection in vitro and three active thiadiazole alkaloids were isolated acting mainly on viral integration and viral entry.

An amplification-free CRISPR-Cas12a assay for titer determination and composition analysis of the rAAV genome

The viral genome titer is a crucial indicator for the clinical dosing, manufacturing, and analytical testing of recombinant adeno-associated virus (rAAV) gene therapy products. Although quantitative PCR and digital PCR are the common methods used for quantifying rAAV genome titer, they are limited by inadequate accuracy and robustness. The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a biosensor is being increasingly used in virus detection; however, there is currently no report on its application in the titer determination of gene therapy products. In the present study, an amplification-free CRISPR-Cas12a assay was developed, optimized, and applied for rAAV genome titer determination. The assay demonstrated high precision and accuracy within the detection range of 4 × 109 and 1011 vg/mL. No significant difference was observed between the Cas12a and qPCR assay results (p﹤0.05, t-test). Moreover, Cas12a exhibited similar activity on both single-stranded and double-stranded DNA substrates. Based on this characteristic, the titers of positive-sense and negative-sense strands were determined separately, which revealed a significant difference between their titers for an in-house reference AAV5-IN. This study presents the inaugural report of a Cas12a assay developed for the titer determination and composition analysis of the rAAV genome.

Cardiomyocyte LGR6 alleviates ferroptosis in diabetic cardiomyopathy via regulating mitochondrial biogenesis

AimsThe majority of people with diabetes are susceptible to cardiac dysfunction and heart failure, and conventional drug therapy cannot correct the progression of diabetic cardiomyopathy. We assessed the potential role and therapeutic value of LGR6 (G protein-coupled receptor containing leucine-rich repeats 6) in diabetic cardiomyopathy. Methods and resultsType 2 diabetes models were established using high-fat diet/streptozotocin-induced diabetes in mice. LGR6 knockout mice were generated. Recombinant adeno-associated virus serotype 9 carrying LGR6 under the cardiac troponin T promoter was injected into diabetic mice. Cardiomyocytes incubated with high glucose (HG) were used to imitate diabetic cardiomyopathy in vitro. The molecular mechanism was explored through RNA sequencing and a chromatin immunoprecipitation assay. We found that LGR6 expression was upregulated in diabetic hearts and HL1 cardiomyocytes treated with HG. The LGR6 knockout aggravated, but cardiomyocyte-specific LGR6 overexpression ameliorated, cardiac dysfunction and remodeling in diabetic mice. Mechanistically, in vivo and in vitro experiments revealed that LGR6 deletion aggravated, whereas LGR6 overexpression alleviated, ferroptosis and disrupted mitochondrial biogenesis by regulating STAT3/Pgc1a signaling. STAT3 inhibition and Pgc1a activation abrogated LGR6 knockout-induced mitochondrial dysfunction and ferroptosis in diabetic mice. In addition, LGR6 activation by recombinant RSPO3 treatment ameliorated cardiac dysfunction, ferroptosis and mitochondrial dysfunction in diabetic mice. ConclusionsWe identified a previously undescribed signaling pathway of the LGR6-STAT3-Pgc1a axis that plays a critical role in ferroptosis and mitochondrial disorders during diabetic cardiomyopathy and provides an option for treatment of diabetic hearts.

Impact of the interaction between herpes simplex virus 1 ICP22 and FACT on viral gene expression and pathogenesis.

Facilitates chromatin transcription (FACT) interacts with nucleosomes to promote gene transcription by regulating the dissociation and reassembly of nucleosomes downstream and upstream of RNA polymerase II (Pol II). A previous study reported that herpes simplex virus 1 (HSV-1) regulatory protein ICP22 interacted with FACT and was required for its recruitment to the viral DNA genome in HSV-1-infected cells. However, the biological importance of interactions between ICP22 and FACT in relation to HSV-1 infection is unclear. Here, we mapped the minimal domain of ICP22 required for its efficient interaction with FACT to a cluster of five basic amino acids in ICP22. A recombinant virus harboring alanine substitutions in this identified cluster led to the decreased accumulation of viral mRNAs from UL54, UL38, and UL44 genes, reduced Pol II occupancy of these genes in MRC-5 cells, and impaired HSV-1 virulence in mice following ocular or intracranial infection. Furthermore, the treatment of mice infected with wild-type HSV-1 with CBL0137, a FACT inhibitor currently being investigated in clinical trials, significantly improved the survival rate of mice. These results suggested that the interaction between ICP22 and FACT was required for efficient HSV-1 gene expression and pathogenicity. Therefore, FACT might be a potential therapeutic target for HSV-1 infection.IMPORTANCEICP22 is a well-known regulatory factor of HSV-1 gene expression, but its mechanism(s) are poorly understood. Although the interaction of FACT with ICP22 was reported previously, its significance in HSV-1 infection is unknown. Given that FACT is involved in gene transcription, it is of interest to investigate this interaction as it relates to HSV-1 gene expression. To determine a direct link between the interaction and HSV-1 infection, we mapped a minimal domain of ICP22 required for its efficient interaction with FACT and generated a recombinant virus carrying mutations in the identified domain. Using the recombinant virus, we obtained evidence suggesting that the interaction between ICP22 and FACT promoted Pol II transcription from HSV-1 genes and viral virulence in mice. In addition, CBL0137, an inhibitor of FACT, effectively protected mice from lethal HSV-1 infection, suggesting FACT might be a potential target for the development of novel anti-HSV drugs.

A recombinant Getah Virus expressing a GFP gene for rapid neutralization testing and antiviral drug screening assay

Getah virus (GETV) is a re-emerging mosquito-borne RNA virus that induces fever, hind limb edema, swollen submandibular lymph nodes, and urticaria in horses. In pigs, the virus often results in stillbirths among pregnant sows, and neurological symptoms leading to death in piglets. Currently, there are no specific treatments or drugs available for GETV infection. The use of reporter viruses to monitor viral replication and spread in real-time within infected cells and animals provides a powerful tool for targeting antiviral drugs throughout the viral life cycle. Their fluorescence-tracked characteristics greatly facilitate virus neutralization tests (VNTs). In this study, we engineered two recombinant viruses by inserting different reporter protein genes at the 3′ end of the structural protein gene, an unreported location that can accommodate exogenous genes. The rGEEiLOV and rGEEGFP viruses demonstrated genetic stability for at least five passages and replicated at a rate similar to that of the parental virus in BHK-21 cells. The rGEEGFP virus facilitated viral neutralization testing. Additionally, we used the reporter virus rGEEGFP to confirm ivermectin, a broad-spectrum antiparasitic agent, as a potential inhibitor of GETV in vitro. Ivermectin appears to inhibit the early replication stages of the virus and can block cell-to-cell viral transmission. In conclusion, rGEEGFP holds significant potential for antiviral screening to identify specific inhibitors against GETV and for use in viral neutralization tests.

Generation of recombinant viruses directly from clinical specimens of COVID-19 patients.

Characterization of the causative agent(s) for infectious diseases helps in implementing effective control measurements, especially in outbreaks. However, the isolation of the agent(s) from clinical specimens is often challenging and time-consuming. In this study, saliva samples from coronavirus disease 2019 patients were directly subjected to purifying viral RNA, synthesizing DNA amplicons for sequencing, and generating recombinant viruses. Utilizing an updated circular polymerase extension reaction method, we successfully generated chimeric SARS-CoV-2 viruses with sufficient in vitro replication capacity and antigenicity. Thus, the recombinant viruses generated in this study were applicable for evaluating the antivirals. Collectively, our developed method facilitates rapid characterization of specimens circulating in hosts, aiding in the establishment of control measurements. Additionally, this approach offers an advanced strategy for controlling other (re-)emerging viral infectious diseases.

Efficacy of commercial recombinant HVT vaccines against a North American clade 2.3.4.4b H5N1 highly pathogenic avian influenza virus in chickens.

The outbreak of clade 2.3.4.4b H5 highly pathogenic avian influenza (HPAI) in North America that started in 2021 has increased interest in applying vaccination as a strategy to help control and prevent the disease in poultry. Two commercially available vaccines based on the recombinant herpes virus of turkeys (rHVT) vector were tested against a recent North American clade 2.3.4.4b H5 HPAI virus isolate: A/turkey/Indiana/22-003707-003/2022 H5N1 in specific pathogen free white leghorn (WL) chickens and commercial broiler chickens. One rHVT-H5 vaccine encodes a hemagglutinin (HA) gene designed by the computationally optimized broadly reactive antigen method (COBRA-HVT vaccine). The other encodes an HA gene of a clade 2.2 virus (2.2-HVT vaccine). There was 100% survival of both chicken types COBRA-HVT vaccinated groups and in the 2.2-HVT vaccinated groups there was 94.8% and 90% survival of the WL and broilers respectively. Compared to the 2.2-HVT vaccinated groups, WL in the COBRA-HVT vaccinated group shed significantly lower mean viral titers by the cloacal route and broilers shed significantly lower titers by the oropharyngeal route than broilers. Virus titers detected in oral and cloacal swabs were otherwise similar among both vaccine groups and chicken types. To assess antibody-based tests to identify birds that have been infected after vaccination (DIVA-VI), sera collected after the challenge were tested with enzyme-linked lectin assay-neuraminidase inhibition (ELLA-NI) for N1 neuraminidase antibody detection and by commercial ELISA for detection of antibodies to the NP protein. As early as 7 days post challenge (DPC) 100% of the chickens were positive by ELLA-NI. ELISA was less sensitive with a maximum of 75% positive at 10DPC in broilers vaccinated with 2.2-HVT. Both vaccines provided protection from challenge to both types of chickens and ELLA-NI was sensitive at identifying antibodies to the challenge virus therefore should be evaluated further for DIVA-VI.

Simultaneous Expression of Different Therapeutic Genes by Infection with Multiple Oncolytic HSV-1 Vectors.

Oncolytic viruses (OVs) are anti-cancer therapeutics combining the selective killing of cancer cells with the triggering of an anti-tumoral immune response. The latter effect can be improved by arming OVs with immunomodulatory factors. Due to the heterogeneity of cancer and the tumor microenvironment, it is anticipated that strategies based on the co-expression of multiple therapeutic molecules that interfere with different features of the target malignancy will be more effective than mono-therapies. Here, we show that (i) the simultaneous expression of different proteins in triple-negative breast cancer (TNBC) cells can be achieved through their infection with a combination of OVs based on herpes simplex virus type 1 (oHSV1), each encoding a single transgene. (ii) The level of expressed proteins is dependent on the number of infectious viral particles utilized to challenge tumor cells. (iii) All recombinant viruses exhibited comparable efficacy in the killing of TNBC cells in single and multiple infections and showed similar kinetics of replication. Overall, our results suggest that a strategy based on co-infection with a panel of oHSV1s may represent a promising combinatorial therapeutic approach for TNBC, as well as for other types of solid tumors, that merits further investigation in more advanced in vitro and in vivo models.

Genetic diversity accelerates canine distemper virus adaptation to ferrets.

RNA viruses adapt rapidly to new host environments by generating highly diverse genome sets, so-called "quasispecies." Minor genetic variants promote their rapid adaptation, allowing for the emergence of drug-resistance or immune-escape mutants. Understanding these adaptation processes is highly relevant to assessing the risk of cross-species transmission and the safety and efficacy of vaccines and antivirals. We hypothesized that genetic memory within a viral genome population facilitates rapid adaptation. To test this, we investigated the adaptation of the Morbillivirus canine distemper virus to ferrets and compared an attenuated, Vero cell-adapted virus isolate with its recombinant derivative over consecutive ferret passages. Although both viruses adapted to the new host, the reduced initial genetic diversity of the recombinant virus resulted in delayed disease onset. The non-recombinant virus gradually increased the frequencies of beneficial mutations already present at very low frequencies in the input virus. In contrast, the recombinant virus first evolved de novo mutations to compensate for the initial fitness impairments. Importantly, while both viruses evolved different sets of mutations, most mutations found in the adapted non-recombinant virus were identical to those found in a previous ferret adaptation experiment with the same isolate, indicating that mutations present at low frequency in the original virus stock serve as genetic memory. An arginine residue at position 519 in the carboxy terminus of the nucleoprotein shared by all adapted viruses was found to contribute to pathogenesis in ferrets. Our work illustrates the importance of genetic diversity for adaptation to new environments and identifies regions with functional relevance.IMPORTANCEWhen viruses encounter a new host, they can rapidly adapt to this host and cause disease. How these adaptation processes occur remains understudied. Morbilliviruses have high clinical and veterinary relevance and are attractive model systems to study these adaptation processes. The canine distemper virus is of particular interest, as it exhibits a broader host range than other morbilliviruses and frequently crosses species barriers. Here, we compared the adaptation of an attenuated virus and its recombinant derivative to that of ferrets. Pre-existing mutations present at low frequency allowed faster adaptation of the non-recombinant virus compared to the recombinant virus. We identified a common point mutation in the nucleoprotein that affected the pathogenesis of both viruses. Our study shows that genetic memory facilitates environmental adaptation and that erasing this genetic memory by genetic engineering results in delayed and different adaptation to new environments, providing an important safety aspect for the generation of live-attenuated vaccines.

Evaluation of a rapid multi-attribute combinatorial high-throughput UV-Vis/DLS/SLS analytical platform for rAAV quantification and characterization

Recombinant adeno-associated virus (rAAV)-based gene therapies are expanding in their application. Despite progress in manufacturing, current analytical methods for product quantification and characterization remain largely unchanged. Although critical for product and process development, in-process testing, and batch release, current analytical methods are labor-intensive, costly, and hampered by extended turnaround times and low throughput. The field requires more efficient, cost-effective analytical techniques capable of handling large sample quantities to accelerate product and process development. Here, we evaluated Stunner from Unchained Labs for quantifying and characterizing rAAVs and compared it with established analytical methods. Stunner is a combinatorial analytic technology platform that interpolates ultraviolet-visible (UV-Vis) absorption with static and dynamic light scattering (SLS/DLS) analysis to determine capsid and genomic titer, empty and full capsid ratio, and assess vector size and polydispersity. The platform offers empirical measurements with minimal sample requirements. Upon testing hundreds of rAAV vectors, comprising various serotypes and transgenes, the data show a strong correlation with established analytical methods and exhibit high reproducibility and repeatability. Some analyses can be applied to in-process samples from different purification stages and processes, fulfilling the demand for rapid, high-throughput analysis during development. In sum, the pipeline presented streamlines small- and large-batch analytics.

Sequencing of Kaposi's Sarcoma Herpesvirus (KSHV) genomes from persons of diverse ethnicities and provenances with KSHV-associated diseases demonstrate multiple infections, novel polymorphisms, and low intra-host variance.

Recently published near full-length KSHV genomes from a Cameroon Kaposi sarcoma case-control study showed strong evidence of viral recombination and mixed infections, but no sequence variations associated with disease. Using the same methodology, an additional 102 KSHV genomes from 76 individuals with KSHV-associated diseases have been sequenced. Diagnoses comprise all KSHV-associated diseases (KAD): Kaposi sarcoma (KS), primary effusion lymphoma (PEL), KSHV-associated large cell lymphoma (KSHV-LCL), a type of multicentric Castleman disease (KSHV-MCD), and KSHV inflammatory cytokine syndrome (KICS). Participants originated from 22 different countries, providing the opportunity to obtain new near full-length sequences of a wide diversity of KSHV genomes. These include near full-length sequence of genomes with KSHV K1 subtypes A, B, C, and F as well as subtype E, for which no full sequence was previously available. High levels of recombination were observed. Fourteen individuals (18%) showed evidence of infection with multiple KSHV variants (from two to four unique genomes). Twenty-six comparisons of sequences, obtained from various sampling sites including PBMC, tissue biopsies, oral fluids, and effusions in the same participants, identified near complete genome conservation between different biological compartments. Polymorphisms were identified in coding and non-coding regions, including indels in the K3 and K15 genes and sequence inversions here reported for the first time. One such polymorphism in KSHV ORF46, specific to the KSHV K1 subtype E2, encoded a mutation in the leucine loop extension of the uracil DNA glycosylase that results in alteration of biochemical functions of this protein. This confirms that KSHV sequence variations can have functional consequences warranting further investigation. This study represents the largest and most diverse analysis of KSHV genome sequences to date among individuals with KAD and provides important new information on global KSHV genomics.

Development of a Cell-Based Reporter Potency Assay for Live Virus Vaccines.

The rapid development of potency assays is critical in the development of life-saving vaccines. The traditional plaque assay or fifty percent tissue culture infectious dose (TCID50) assay used to measure the potency of live virus vaccines is time consuming, labor intensive, low throughput and with high variability. Described here is the development and qualification of a cell-based reporter potency assay for two vaccines for respiratory viral infection, one based on the recombinant vesicular stomatitis virus (rVSV) backbone, termed Vaccine 1 in this paper, and the other based on the measles virus vector, termed Vaccine 2. The reporter potency assay used a Vero E6 cell line engineered to constitutively express NanuLuc® luciferase, termed the VeroE6-NLuc or JM-1 cell line. Infection of JM-1 cells by a live virus, such as rVSV or measles virus, causes a cytopathic effect (CPE) and release of NanuLuc® from the cytoplasm into the supernatant, the amount of which reflects the intensity of the viral infection. The relative potency was calculated by comparison to a reference standard using parallel line analysis (PLA) in a log-log linear model. The reporter assay demonstrated good linearity, accuracy, and precision, and is therefore suitable for a vaccine potency assay. Further evaluation of the Vaccine 1 reporter assay demonstrated the robustness to a range of deliberate variation of the selected assay parameters and correlation with the plaque assay. In conclusion, we have demonstrated that the reporter assay using the JM-1 cell line could be used as a potency assay to support the manufacturing and release of multiple live virus vaccines.

Distinct pathway for evolution of enhanced receptor binding and cell entry in SARS-like bat coronaviruses.

Understanding the zoonotic risks posed by bat coronaviruses (CoVs) is critical for pandemic preparedness. Herein, we generated recombinant vesicular stomatitis viruses (rVSVs) bearing spikes from divergent bat CoVs to investigate their cell entry mechanisms. Unexpectedly, the successful recovery of rVSVs bearing the spike from SHC014, a SARS-like bat CoV, was associated with the acquisition of a novel substitution in the S2 fusion peptide-proximal region (FPPR). This substitution enhanced viral entry in both VSV and coronavirus contexts by increasing the availability of the spike receptor-binding domain to recognize its cellular receptor, ACE2. A second substitution in the spike N-terminal domain, uncovered through forward-genetic selection, interacted epistatically with the FPPR substitution to synergistically enhance spike:ACE2 interaction and viral entry. Our findings identify genetic pathways for adaptation by bat CoVs during spillover and host-to-host transmission, fitness trade-offs inherent to these pathways, and potential Achilles' heels that could be targeted with countermeasures.

High throughput profiling identified PA-L106R amino acid substitution in A(H1N1)pdm09 influenza virus that confers reduced susceptibility to baloxavir in vitro

Baloxavir acid (BXA) is a pan-influenza antiviral that targets the cap-dependent endonuclease of the polymerase acidic (PA) protein required for viral mRNA synthesis. To gain a comprehensive understanding on the molecular changes associated with reduced susceptibility to BXA and their fitness profile, we performed a deep mutational scanning at the PA endonuclease domain of an A (H1N1)pdm09 virus. The recombinant virus libraries were serially passaged in vitro under increasing concentrations of BXA followed by next-generation sequencing to monitor PA amino acid substitutions with increased detection frequencies. Enriched PA amino acid changes were each introduced into a recombinant A (H1N1)pdm09 virus to validate their effect on BXA susceptibility and viral replication fitness in vitro. The I38 T/M substitutions known to confer reduced susceptibility to BXA were invariably detected from recombinant virus libraries within 5 serial passages. In addition, we identified a novel L106R substitution that emerged in the third passage and conferred greater than 10-fold reduced susceptibility to BXA. PA-L106 is highly conserved among seasonal influenza A and B viruses. Compared to the wild-type virus, the L106R substitution resulted in reduced polymerase activity and a minor reduction of the peak viral load, suggesting the amino acid change may result in moderate fitness loss. Our results support the use of deep mutational scanning as a practical tool to elucidate genotype-phenotype relationships, including mapping amino acid substitutions with reduced susceptibility to antivirals.

Adeno-associated virus delivered CXCL9 sensitizes glioblastoma to anti-PD-1 immune checkpoint blockade

There are numerous mechanisms by which glioblastoma cells evade immunological detection, underscoring the need for strategic combinatorial treatments to achieve appreciable therapeutic effects. However, developing combination therapies is difficult due to dose-limiting toxicities, blood-brain-barrier, and suppressive tumor microenvironment. Glioblastoma is notoriously devoid of lymphocytes driven in part by a paucity of lymphocyte trafficking factors necessary to prompt their recruitment and activation. Herein, we develop a recombinant adeno-associated virus (AAV) gene therapy that enables focal and stable reconstitution of the tumor microenvironment with C-X-C motif ligand 9 (CXCL9), a powerful call-and-receive chemokine for lymphocytes. By manipulating local chemokine directional guidance, AAV-CXCL9 increases tumor infiltration by cytotoxic lymphocytes, sensitizing glioblastoma to anti-PD-1 immune checkpoint blockade in female preclinical tumor models. These effects are accompanied by immunologic signatures evocative of an inflamed tumor microenvironment. These findings support AAV gene therapy as an adjuvant for reconditioning glioblastoma immunogenicity given its safety profile, tropism, modularity, and off-the-shelf capability.

Detection of transgenes in equine dried blood spots using digital PCR and qPCR for gene doping control.

Due to the ease of collection, transport and storage, the use of dried blood spots (DBS) offers an attractive alternative matrix for detection of the abuse of gene therapy, otherwise known as gene doping. This study evaluated the recovery, extraction efficiency and resulting detection capability of DNA from DBS by evaluating different target types, DNA extraction kits, the number of punches and blood tube preservatives. The long-term storage stability of low-copy-number transgene targets in DBS was not assessed in this study but would be noteworthy to investigate further. DNA was quantified using two detection methods: qPCR and digital PCR (dPCR). Using six punches with the Qiagen Investigator kit gave the best overall DNA yield compared with other extraction methods. Including three punches, however, gave better DNA extraction efficiency. Reference material could be detected using qPCR and dPCR in DBS spiked with 5000 copies/mL of blood (approximately 15 copies per 3 mm of punch). The optimal DNA extraction protocol was used on DBS samples from a custom recombinant adeno-associated virus administration study and showed successful detection of vector targets in DBS samples.

Recombinant Adeno-Associated Virus Vectors for Gene Therapy of the Central Nervous System: Delivery Routes and Clinical Aspects.

The Central Nervous System (CNS) is vulnerable to a range of diseases, including neurodegenerative and oncological conditions, which present significant treatment challenges. The blood-brain barrier (BBB) restricts molecule penetration, complicating the achievement of therapeutic concentrations in the CNS following systemic administration. Gene therapy using recombinant adeno-associated virus (rAAV) vectors emerges as a promising strategy for treating CNS diseases, demonstrated by the registration of six gene therapy products in the past six years and 87 ongoing clinical trials. This review explores the implementation of rAAV vectors in CNS disease treatment, emphasizing AAV biology and vector engineering. Various administration methods-such as intravenous, intrathecal, and intraparenchymal routes-and experimental approaches like intranasal and intramuscular administration are evaluated, discussing their advantages and limitations in different CNS contexts. Additionally, the review underscores the importance of optimizing therapeutic efficacy through the pharmacokinetics (PK) and pharmacodynamics (PD) of rAAV vectors. A comprehensive analysis of clinical trials reveals successes and challenges, including barriers to commercialization. This review provides insights into therapeutic strategies using rAAV vectors in neurological diseases and identifies areas requiring further research, particularly in optimizing rAAV PK/PD.

Sleeping Beauty mRNA-LNP enables stable rAAV transgene expression in mouse and NHP hepatocytes and improves vector potency

Recombinant adeno-associated virus (rAAV) vector gene delivery systems have demonstrated great promise in clinical trials but continue to face durability and dose-related challenges. Unlike rAAV gene therapy, integrating gene addition approaches can provide curative expression in mitotically active cells and pediatric populations. We explored a novel invivo delivery approach based on an engineered transposase, Sleeping Beauty (SB100X), delivered as an mRNA within a lipid nanoparticle (LNP), in combination with an rAAV-delivered transposable transgene. This combinatorial approach achieved correction of ornithine transcarbamylase deficiency in the neonatal Spfash mouse model following a single delivery to dividing hepatocytes in the newborn liver. Correction remained stable into adulthood, while a conventional rAAV approach resulted in a return to the disease state. In non-human primates, integration by transposition, mediated by this technology, improved gene expression 10-fold over conventional rAAV-mediated gene transfer while requiring 5-fold less vector. Additionally, integration site analysis confirmed a random profile while specifically targeting TA dinucleotides across the genome. Together, these findings demonstrate that transposable elements can improve rAAV-delivered therapies by lowering the vector dose requirement and associated toxicity while expanding target cell types.

Safety and Immunogenicity of Morbillivirus canis Vaccines for Domestic and Wild Animals: A Scoping Review.

Morbillivirus canis (canine distemper virus (CDV)) is recognized as a multihost pathogen responsible for a transmissible disease affecting both domestic and wild animals. A considerable portion of wildlife populations remain unvaccinated due to a lack of safety and immunogenicity data on existing vaccines for the prevention of CDV infection in these species. This review aimed to assess the current state of CDV vaccination research for both domestic and wild animals and to explore novel vaccine candidates through in vivo studies. It also sought to synthesize the scattered information from the extensive scientific literature on CDV vaccine research, identify key researchers in the field, and highlight areas where research on CDV vaccination is lacking. A scoping review was conducted across four databases following the PRISMA-ScR protocol, with information analyzed using absolute and relative frequencies and 95% confidence intervals (CIs) for study number proportions. Among the 2321 articles retrieved, 68 met the inclusion criteria and focused on CDV vaccines in various animal species, such as dogs, ferrets, minks, and mice. Most of the scientific community involved in this research was in the USA, Canada, France, and Denmark. Various vaccine types, including MLV CDV, recombinant virus, DNA plasmids, inactivated CDV, and MLV measles virus (MeV), were identified, along with diverse immunization routes and schedules employed in experimental and commercial vaccines. Safety and efficacy data were summarized. Notably, 37 studies reported postimmunization CDV challenge, primarily in dogs, revealing the survival rates of vaccinated animals. In summary, CDV vaccines generally demonstrate an acceptable safety profile in dogs and show promise as a means of controlling CDV. However, significant gaps in vaccine research persist, particularly concerning wildlife reservoirs, indicating the need for further investigation.