Optimizing Transfection Efficiency of Spermine Polar Head Cholesterol-based Cationic Lipids with Amino Acid Linker.

In this work, a series of spermine polar head cholesterol-based cationic lipids with various amino acid spacers were synthesized and evaluated as non-viral gene delivery systems. The physicochemical properties of the resulting lipoplexes, formed from these lipids and DOPE, were assessed, including zeta-potential, DNA binding and DNA protection from serum. Transfection efficiency and cytotoxicity were examined under serum-free and 10-40% serum-containing conditions. The results showed that the physicochemical properties of cationic lipids, both with and without amino acid spacers, were not significantly different. Cationic liposomes composed of lipid Sper-Ahx-Chol, which has a 6-aminohexanoic acid spacer, and DOPE exhibited greater transfection efficiency in HeLa cells compared to Lipofectamine3000, both in the absence and presence of 10-40% serum. Additionally, lipid Sper-His-Chol with a histidine spacer and Sper-Ahx-Chol showed higher efficiency than Lipofectamine3000 against HEK293T under 40% serum conditions. These results suggest that the incorporation of amino acids into the cationic lipids can significantly enhance their DNA delivery efficiency. Specifically, certain amino acid modifications improved transfection efficiency while maintaining low cytotoxicity. Our findings highlight the potential of amino acid-tailored cationic lipids as promising vectors for enhanced DNA delivery.

Laminar specificity and coverage of viral-mediated gene expression restricted to GABAergic interneurons and their parvalbumin subclass in marmoset primary visual cortex.

In the mammalian neocortex, inhibition is important for dynamically balancing excitation and shaping the response properties of cells and circuits. The various computational functions of inhibition are thought to be mediated by different inhibitory neuron types, of which a large diversity exists in several species. Current understanding of the function and connectivity of distinct inhibitory neuron types has mainly derived from studies in transgenic mice. However, it is unknown whether knowledge gained from mouse studies applies to the non-human primate, the model system closest to humans. The lack of viral tools to selectively access inhibitory neuron types has been a major impediment to studying their function in the primate. Here, we have thoroughly validated and characterized several recently developed viral vectors designed to restrict transgene expression to GABAergic cells or their parvalbumin (PV) subtype, and identified two types that show high specificity and efficiency in marmoset V1. We show that in marmoset V1, AAV-h56D induces transgene expression in GABAergic cells with up to 91-94% specificity and 79% efficiency, but this depends on viral serotype and cortical layer. AAV-PHP.eB-S5E2 induces transgene expression in PV cells across all cortical layers with up to 98% specificity and 86-90% efficiency, depending on layer. Thus, these viral vectors are promising tools for studying GABA and PV cell function and connectivity in the primate cortex.

Advances in Nanoparticles as Non-Viral Vectors for Efficient Delivery of CRISPR/Cas9.

The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system is a gene-editing technology. Nanoparticle delivery systems have attracted attention because of the limitations of conventional viral vectors. In this review, we assess the efficiency of various nanoparticles, including lipid-based, polymer-based, inorganic, and extracellular vesicle-based systems, as non-viral vectors for CRISPR/Cas9 delivery. We discuss their advantages, limitations, and current challenges. By summarizing recent advancements and highlighting key strategies, this review aims to provide a comprehensive overview of the role of non-viral delivery systems in advancing CRISPR/Cas9 technology for clinical applications and gene therapy.

Tuning VSV-G Expression Improves Baculovirus Integrity, Stability and Mammalian Cell Transduction Efficiency.

Baculoviral vectors (BVs) derived from Autographa californica multiple nucleopolyhedrovirus (AcMNPV) are an attractive tool for multigene delivery in mammalian cells, which is particularly relevant for CRISPR technologies. Most applications in mammalian cells rely on BVs that are pseudotyped with vesicular stomatitis virus G-protein (VSV-G) to promote efficient endosomal release. VSV-G expression typically occurs under the control of the hyperactive polH promoter. In this study, we demonstrate that polH-driven VSV-G expression results in BVs characterised by reduced stability, impaired morphology, and VSV-G induced toxicity at high multiplicities of transduction (MOTs) in target mammalian cells. To overcome these drawbacks, we explored five alternative viral promoters with the aim of optimising VSV-G levels displayed on the pseudotyped BVs. We report that Orf-13 and Orf-81 promoters reduce VSV-G expression to less than 5% of polH, rescuing BV morphology and stability. In a panel of human cell lines, we elucidate that BVs with reduced VSV-G support efficient gene delivery and CRISPR-mediated gene editing, at levels comparable to those obtained previously with polH VSV-G-pseudotyped BVs (polH VSV-G BV). These results demonstrate that VSV-G hyperexpression is not required for efficient transduction of mammalian cells. By contrast, reduced VSV-G expression confers similar transduction dynamics while substantially improving BV integrity, structure, and stability.

Retinal damage promotes mitochondrial transfer in the visual system of a mouse model of Leber hereditary optic neuropathy.

Lenadogene nolparvovec is a gene therapy which has been developed to treat Leber hereditary optic neuropathy (LHON) caused by a point mutation in the mitochondrial NADH dehydrogenase 4 (ND4) gene. Clinical trials have demonstrated a significant improvement of visual acuity up to 5 years after treatment by lenadogene nolparvovec but, surprisingly, unilateral treatment resulted in bilateral improvement of vision. This contralateral effect - similarly observed with other gene therapy products in development for MT-ND4-LHON - is supported by the migration of viral vector genomes and their transcripts to the contralateral eye, as reported in animals, and post-mortem samples from two patients. In this study, we used an AAV2 encoding fluorescent proteins targeting mitochondria to investigate whether these organelles themselves could transfer from the treated eye to the fellow one. We found that mitochondria travel along the visual system (optic chiasm and primary visual cortex) and reach the contralateral eye (optic nerve and retina) in physiological conditions. We also observed that, in a rotenone-induced model of retinal damage mimicking LHON, mitochondrial transfer from the healthy to the damaged eye was accelerated and enhanced. Our results thus provide a further explanation for the contralateral beneficial effect observed during clinical studies with lenadogene nolparvovec.

Gene therapy for the leukodystrophies: From preclinical animal studies to clinical trials.

Leukodystrophies are progressive single gene disorders affecting the white matter of the brain. Several gene therapy trials are in progress to address the urgent unmet need for this patient population. We performed a comprehensive literature review of all gene therapy clinical trials listed in www.clinicaltrials.gov through August 2024, and the relevant preclinical studies that enabled clinical translation. Of the approximately 50 leukodystrophies described to date, only eight have existing gene therapy clinical trials: metachromatic leukodystrophy, X-linked adrenoleukodystrophy, globoid cell leukodystrophy, Canavan disease, giant axonal neuropathy, GM2 gangliosidoses, Alexander disease and Pelizaeus-Merzbacher disease. What led to the emergence of gene therapy trials for these specific disorders? What preclinical data or disease context was enabling? For each of these eight disorders, we first describe its pathophysiology and clinical presentation. We discuss the impact of gene therapy delivery route, targeted cell type, delivery modality, dosage, and timing on therapeutic efficacy. We note that use of allogeneic hematopoietic stem cell transplantation in some leukodystrophies allowed for an accelerated path to clinic even in the absence of available animal models. In other leukodystrophies, small and large animal model studies enabled clinical translation of experimental gene therapies. Human clinical trials for the leukodystrophies include ex vivo lentiviral gene delivery, in vivo AAV-mediated gene delivery, and intrathecal antisense oligonucleotide approaches. We outline adverse events associated with each modality focusing specifically on genotoxicity and immunotoxicity. We review monitoring and management of events related to insertional mutagenesis and immune responses. The data presented in this review show that gene therapy, while promising, requires systematic monitoring to account for the precarious disease biology and the adverse events associated with new technology.

Safety pharmacology of human endogenous retrovirus-enveloped baculoviral DNA vaccines against SARS-CoV-2 in Sprague-Dawley rats and beagle dogs.

The coronavirus disease 2019 (COVID-19) emerged as a major global health crisis, posing significant health, economic, and social challenges. Vaccine development has been a crucial response to the severe-acute-respiratory-syndrome-related coronavirus-2 pandemic owing to the critical role of immunization in controlling infectious diseases, leading to the expedited development of several effective vaccines. Although mRNA platform-based COVID-19 vaccines authorized under emergency-use authorization have been administered globally, concerns regarding the vaccines have increased owing to the occurrence of various side effects. The present study aimed to evaluate the safety of a non-replicating recombinant baculovirus expressing the human endogenous retrovirus envelope gene (AcHERV) vaccine encoding SARS-CoV-2 antigens. Owing to the limited number of existing safety pharmacology studies on AcHERV as a viral vector vaccine, we conducted neurobehavior (Modified Irwin's Test), body temperature, and respiratory function studies in rats and cardiovascular system studies in male beagle dogs, which were administered the AcHERV-COVID-19 vaccine using telemetry. The safety assessment revealed no significant toxicological alterations. However, in rats, both sexes administered with the AcHERV-COVID-19 vaccine exhibited a temporary increase in body temperature, which normalized or showed signs of recovery. In conclusion, AcHERV-COVID-19 demonstrates a sufficient safety profile that supports its potential evaluation in future clinical trials.

Fed-batch strategies for intensified rVSV vector production in high cell density cultures of suspension HEK293 cells.

Vesicular stomatitis virus (VSV) has been increasingly demonstrated as a promising viral vector platform. As the interest over this modality for vaccine and gene therapy applications increases, the need for intensified processes to produce these vectors emerge. In this study, we develop fed-batch-based operations to intensify the production of a recombinant VSV-based vaccine candidate (rVSV-SARS-CoV-2) in suspension cultures of HEK293 cells. A feeding strategy, in which a commercial concentrated medium was added to cultures based on cell growth through a fixed cell specific feeding rate (CSFR), was applied for the development of two different processes using Ambr250 modular bioreactors. Cultures operated in hybrid fed-batch/perfusion (FB/P) or fed-batch (FB) were able to sustain infections performed at 8.0 × 106 cells/mL, respectively resulting in 3.9 and 5.0-fold increase in total yield (YT) and 1.7 and 5.6-fold increase in volumetric productivity (VP) when compared with a batch reference. A maximum viral titer of 4.5 × 1010 TCID50/mL was reached, which is comparable or higher than other processes for VSV production in different cell lines. Overall, our study reports efficient fed-batch options to intensify the production of a rVSV-based vaccine candidate in suspension HEK293 cells.

Development of a recombinant non-replicating Newcastle disease virus.

RESEARCH HIGHLIGHTS: Development of nr-NDV.Reverse transfection was applied for the recovery of nr-NDV.Propagation of nr-NDV was done by sub-passaging transfected BSR T7/5 cells.Safety profile was done to prove that the nr-NDV is non-replicating.

Novel recombinant vaccinia virus-vectored vaccine affords complete protection against homologous Borrelia burgdorferi infection in mice.

The rising prevalence of Lyme disease (LD) in North America and Europe has emerged as a pressing public health concern. Despite the availability of veterinary LD vaccines, no vaccine is currently available for human use. Outer surface protein C (OspC) found on the outer membrane of the causative agent, Borrelia burgdorferi, has been identified as a promising target for LD vaccine development due to its sustained expression during mammalian infection. However, the efficacy and immunological mechanisms of LD vaccines solely targeting OspC are not well characterized. In this study, we developed an attenuated Vaccinia virus (VV) vectored vaccine encoding type A OspC (VV-OspC-A). Two doses of the VV-OspC-A vaccine conferred complete protection against homologous B. burgdorferi challenge in mice. Furthermore, the candidate vaccine also prevented the development of carditis and lymph node hyperplasia associated with LD. When investigating the humoral immune response to vaccination, VV-OspC-A was found to induce a robust antibody response predominated by the IgG2a subtype, indicating a Th1-bias. Using a novel quantitative flow cytometry assay, we also determined that elicited antibodies were capable of inducing antibody-dependent cellular phagocytosis in vitro. Finally, we demonstrated that VV-OspC-A vaccination generated a strong antigen-specific CD4+ T-cell response characterized by the secretion of numerous cytokines upon stimulation of splenocytes with OspC peptides. This study suggests a promising avenue for LD vaccine development utilizing viral vectors targeting OspC and provides insights into the immunological mechanisms that confer protection against B. burgdorferi infection.

Targeted gene therapy for rare genetic kidney diseases.

Chronic kidney disease (CKD) is one of the leading causes of mortality worldwide because of kidney failure and the associated challenges of its treatment including dialysis and kidney transplantation. About one-third of CKD cases are linked to inherited monogenic factors, making them suitable for potential gene therapy interventions. However, the intricate anatomical structure of the kidney poses a challenge, limiting the effectiveness of targeted gene delivery to the renal system. In this review, we explore the progress made in the field of targeted gene therapy approaches and their implications for rare genetic kidney disorders, examining preclinical studies and prospects for clinical application. In vivo gene therapy is most commonly used for kidney-targeted gene delivery and involves administering viral and non-viral vectors through various routes such as systemic, renal vein and renal arterial injections. Small nucleic acids have also been used in preclinical and clinical studies for treating certain kidney disorders. Unexpectedly, hematopoietic stem and progenitor cells have been used as an ex vivo gene therapy vehicle for kidney gene delivery, highlighting their ability to differentiate into macrophages within the kidney, forming tunneling nanotubes that can deliver genetic material and organelles to adjacent kidney cells, even across the basement membrane to target the proximal tubular cells. As gene therapy technologies continue to advance and our understanding of kidney biology deepens, there is hope for patients with genetic kidney disorders to eventually avoid kidney transplantation.

Revolutionizing Veterinary Health with Viral Vector-Based Vaccines.

Vaccines signify one of the economical and reasonable means to prevent and eradicate the important infectious diseases. Conventional vaccines like live attenuated and inactivated vaccines comprise of whole pathogen either in attenuated or killed form. While, new generation vaccines have been designed to elicit immune response by genetically modifying only the nucleic acid portion of that pathogen. These new generation therapeutics include mRNA vaccines, DNA plasmid vaccines, chimeric vaccines and recombinant viral vector-based vaccines. Nucleic acid based vaccines use genetic material itself thus, they are highly stable and potent in nature to induce long-lasting immune response. Amongst these novel vaccine platforms, viral vector-based vaccines is one such emerging field which has proven to be extremely effective and potent. Nowadays, veterinary medicine has also accepted this innovative vectored vaccine platform to develop an effective control strategy against certain important viral diseases of animals. Viral vector-based vaccine uses various DNA and RNA viruses of human or animal origin to carry an immunogenic transgene of target pathogen. These vaccines enhance both humoral and cell mediated immune response without use of any accessory immune-stimulants. Till today, several viruses have been modified to be characterized as vaccine vectors. Currently, large number of research programs are going on to develop vectored vaccines and novel viral vector for veterinary use. In the present review, different kinds of viral vectored vaccines having veterinary importance have been discussed.

Characterization of AAV vectors: A review of analytical techniques and critical quality attributes.

Standardized evaluation of adeno-associated virus (AAV) vector products for biotherapeutic application is essential to ensure the safety and efficacy of gene therapies. This includes analyzing the critical quality attributes of the product. However, many of the current analytical techniques used to assess these attributes have limitations, including low throughput, large sample requirements, poorly understood measurement variability, and lack of comparability between methods. To address these challenges, it is essential to establish higher-order reference methods that can be used for comparability measurements, optimization of current assays, and development of reference materials. Highly precise methods are necessary for measuring the empty/partial/full capsid ratios and the titer of AAV vectors. Additionally, it is important to develop methods for the measurement of less-established critical quality attributes, including post-translational modifications, capsid stoichiometry, and methylation profiles. By doing so, we can gain a better understanding of the influence of these attributes on the quality of the product. Moreover, quantification of impurities, such as host-cell proteins and DNA contaminants, is crucial for obtaining regulatory approval. The development and application of refined methodologies will be essential to thoroughly characterize AAV vectors by informing process development and facilitating the generation of reference materials for assay validation and calibration.

Advances in CRISPR/Cas systems-based cell and gene therapy.

Cell and gene therapy are innovative biomedical strategies aimed at addressing diseases at their genetic origins. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) systems have become a groundbreaking tool in cell and gene therapy, offering unprecedented precision and versatility in genome editing. This chapter explores the role of CRISPR in gene editing, tracing its historical development and discussing biomolecular formats such as plasmid, RNA, and protein-based approaches. Next, we discuss CRISPR delivery methods, including viral and non-viral vectors, followed by examining the various engineered CRISPR variants for their potential in gene therapy. Finally, we outline emerging clinical applications, highlighting the advancements in CRISPR for breakthrough medical treatments.

Gene therapy for atrial fibrillation.

Atrial fibrillation (AF) is the most common sustained arrhythmia in adults. Current limitations of pharmacological and ablative therapies motivate the development of novel therapies as next generation treatments for AF. The arrhythmia mechanisms creating and sustaining AF are key elements in the development of this novel treatment. Gene therapy provides a useful platform that allows us to regulate the mechanisms of interest using a suitable transgene(s), vector, and delivery method. Effective gene therapy strategies in the literature have targeted maladaptive electrical or structural remodeling that increase vulnerability to AF. In this review, we will summarize key elements of gene therapy for AF, including molecular targets, gene transfer vectors, atrial gene delivery and preclinical efficacy and toxicity testing. Recent advances and challenges in the field will be also discussed.

Investigating the effect of reduced temperatures on the efficacy of rhabdovirus-based viral vector platforms.

Rhabdoviral vectors can induce lysis of cancer cells. While studied almost exclusively at 37 °C, viruses are subject to a range of temperatures in vivo, including temperatures ≤31 °C. Despite potential implications, the effect of temperatures <37 °C on the performance of rhabdoviral vectors is unknown. We investigated the effect of low anatomical temperatures on two rhabdoviruses, vesicular stomatitis virus (VSV) and Maraba virus (MG1). Using a metabolic resazurin assay, VSV- and MG1-mediated oncolysis was characterized in a panel of cell lines at 28, 31, 34 and 37 °C. The oncolytic ability of both viruses was hindered at 31 and 28 °C. Cold adaptation of both viruses was attempted as a mitigation strategy. Viruses were serially passaged at decreasing temperatures in an attempt to induce mutations. Unfortunately, the cold-adaptation strategies failed to potentiate the oncolytic activity of the viruses at temperatures <37 °C. Interestingly, we discovered that viral replication was unaffected at low temperatures despite the abrogation of oncolytic activity. In contrast, the proliferation of cancer cells was reduced at low temperatures. Equivalent oncolytic effects could be achieved if cells at low temperatures were treated with viruses for longer times. This suggests that rhabdovirus-mediated oncolysis could be compromised at low temperatures in vivo where therapeutic windows are limited.

Viral vector- and virus-like particle-based vaccines against infectious diseases: A minireview.

To overcome the limitations of conventional vaccines, new platforms for vaccine design have emerged such as those based on viral vectors and virus-like particles (VLPs). Viral vector vaccines are highly efficient and the onset of protection is quick. Many recombinant vaccine candidates for humans are based on viruses belonging to different families such as Adenoviridae, Retroviridae, Paramyxoviridae, Rhabdoviridae, and Parvoviridae. Also, the first viral vector vaccine licensed for human vaccination was the Japanese encephalitis virus vaccine. Since then, several viral vectors have been approved for vaccination against the viruses of Lassa fever, Ebola, hepatitis B, hepatitis E, SARS-CoV-2, and malaria. VLPs are nanoparticles that mimic viral particles formed from the self-assembly of structural proteins and VLP-based vaccines against hepatitis B and E viruses, human papillomavirus, and malaria have been commercialized. As evidenced by the accelerated production of vaccines against COVID-19, these new approaches are important tools for vaccinology and for generating rapid responses against pathogens and emerging pandemic threats.

Gene therapy for ultrarare diseases: a geneticist's perspective.

Gene therapy has made considerable strides in recent years. More than 4000 protein-coding genes have been implicated in more than 6000 genetic diseases; next-generation sequencing has dramatically revolutionized the diagnosis of genetic diseases. Most genetic diseases are considered very rare or ultrarare, defined here as having fewer than 1:100,000 cases, but only one of the 12 approved gene therapies (excluding RNA therapies) targets an ultrarare disease. This article explores three gene supplementation therapy approaches suitable for various rare genetic diseases: lentiviral vector-modified autologous CD34+ hematopoietic stem cell transplantation, systemic delivery of adeno-associated virus (AAV) vectors to the liver, and local AAV delivery to the cerebrospinal fluid and brain. Together with RNA therapies, we propose a potential business model for these gene therapies.

Quantifying Dendritic Arbors In Vitro and In Vivo in Rodent Models.

Dendritic arborization is a critical determinant of neuronal connectivity. The structure of a neuron's dendritic arbor determines the number of synaptic inputs a neuron can receive and how it processes synaptic input from other neurons. Here, we describe methods for visualizing and quantifying the dendritic arbor in primary cell cultures and in the intact rodent brain. These techniques can be used to answer significant scientific questions, such as the effects of disease processes, drugs, growth factors, and diverse environmental stressors on dendritogenesis in both in vitro and in vivo rodent models.

Vaccination of cattle with a virus vector vaccine against a major membrane protein of Mycobacterium avium subsp. paratuberculosis elicits CD8 cytotoxic T cells that kill intracellular bacteria.

Analysis of the recall response ex vivo in cattle vaccinated with a Mycobacterium avium subsp. paratuberculosis (Map) rel deletion mutant revealed the immune response was directed toward a 35 kD major membrane protein (MMP) of Map. Antigen presenting cells (APC) primed with MMP elicited expansion of CD8 cytotoxic memory T cells (CTL) with ability to kill intracellular bacteria. Development of CTL was MHC-restricted. The gene MAP2121c, encoding MMP, was modified for expression of MMP (tPA-MMP-2mut) in a mammalian cell line to explore the potential of developing MMP as a vaccine. Ex vivo stimulation of PBMC, from Map free cattle, with APC primed with tPA-MMP-2mut expressed p35 elicited a primary CD8 CTL response comparable to the recall response elicited with PBMC from cattle vaccinated with either the Maprel deletion mutant or MMP. In the present study, the modified gene for MMP, now referred to as p35NN, was placed into a bovine herpes virus-4 (BoHV4) vector to determine the potential use of BoHV-4AΔTK-p35NN as a peptide-based vaccine. Subcutaneous vaccination of healthy cattle with BoHV-4AΔTK-p35NN elicited a CTL recall response, as detected ex vivo. The results show use of a virus vector is an effective way for delivery of MMP as a vaccine. The immunogenic activity of MMP was not lost when modified for expression in mammalian cells. The next step is to conduct a field trial to determine if presence of an immune response to MMP prevents Map from establishing an infection.