Efficient Gene Transfer Research Articles

Comprehensive analysis on Wnt signaling pathway to develop a novel signal modifier

Wnt signals play a key role in organ formation and function. Abnormalities in organ function or genetic diseases and carcinogenesis could point to issues with these signals, they are therefore an effective target for drug delivery. Dr Yuki Ikebuchi, Department of Pharmacy, The University of Tokyo Hospital, Japan, is heading up a team of researchers working to shed greater light on the Wnt signalling pathway. This will facilitate the development and delivery of more targeted drug therapies, helping patients. One element of Ikebuchi’s research is a detailed analysis on the Wnt signalling pathway to develop a novel signal modifier. As previous studies have found that Wnt molecules exert their physiological activities through glycosylation and lipid modification, a possible method to input signals downstream of the Wnt-Fzd complex is to crosslink and interact with Fzd molecules or co-receptors using antibody-like molecules. The researchers sought to obtain high-definition signal activation profiles of 10 types of Fzd molecules; something that hasnâ–™t been done before. At first, the team established the cell line, derived from mouse osteoblast-like MC3T3-E1 cells, in which endogenously expressed all Fzd and co-receptor genese were knocked out using CRISPR-Cas9 system. And then, they used adenoviruses containing the gene sequences of Wnt, Fzd and the co-receptor to transiently express them in knocked-out cells. The researchers will assess the resulting activation of downstream pathways by Luciferase assays using various reporter sequences. They will adopt a dual-glo assay that can correct the gene transfer efficiency and further enhance accuracy using NanoLuc.

Plasmid-free production of the plant lignan pinoresinol in growing Escherichia coli cells

BackgroundThe high-value aryl tetralin lignan (+)-pinoresinol is the main precursor of many plant lignans including (-)-podophyllotoxin, which is used for the synthesis of chemotherapeutics. As (-)-podophyllotoxin is traditionally isolated from endangered and therefore limited natural sources, there is a particular need for biotechnological production. Recently, we developed a reconstituted biosynthetic pathway from (+)-pinoresinol to (-)-deoxypodophyllotoxin, the direct precursor of (-)-podophyllotoxin, in the recombinant host Escherichia coli. However, the use of the expensive substrate (+)-pinoresinol limits its application from the economic viewpoint. In addition, the simultaneous expression of multiple heterologous genes from different plasmids for a multi-enzyme cascade can be challenging and limits large-scale use.ResultsIn this study, recombinant plasmid-free E. coli strains for the multi-step synthesis of pinoresinol from ferulic acid were constructed. To this end, a simple and versatile plasmid toolbox for CRISPR/Cas9-assisted chromosomal integration has been developed, which allows the easy transfer of genes from the pET vector series into the E. coli chromosome. Two versions of the developed toolbox enable the efficient integration of either one or two genes into intergenic high expression loci in both E. coli K-12 and B strains. After evaluation of this toolbox using the fluorescent reporter mCherry, genes from Petroselinum crispum and Zea mays for the synthesis of the monolignol coniferyl alcohol were integrated into different E. coli strains. The product titers achieved with plasmid-free E. coli W3110(T7) were comparable to those of the plasmid-based expression system. For the subsequent oxidative coupling of coniferyl alcohol to pinoresinol, a laccase from Corynebacterium glutamicum was selected. Testing of different culture media as well as optimization of gene copy number and copper availability for laccase activity resulted in the synthesis of 100 mg/L pinoresinol using growing E. coli cells.ConclusionsFor efficient and simple transfer of genes from pET vectors into the E. coli chromosome, an easy-to-handle molecular toolbox was developed and successfully tested on several E. coli strains. By combining heterologous and endogenous enzymes of the host, a plasmid-free recombinant E. coli growing cell system has been established that enables the synthesis of the key lignan pinoresinol.

Development of Mesenchymal Stem Cell Encoded with Myogenic Gene for Treating Radiation-Induced Muscle Fibrosis.

Radiation therapy (RT) is a typical treatment for head and neck cancers. However, prolonged irradiation of the esophagus can cause esophageal fibrosis due to increased reactive oxygen species and proinflammatory cytokines. The objective of this study was to determine whether myogenic gene-transfected mesenchymal stem cells (MSCs) could ameliorate damage to esophageal muscles in a mouse model of radiation-induced esophageal fibrosis. We cloned esophageal myogenic genes (MyoD, MyoG, and Myf6) using plasmid DNA. Afterward, myogenic genes were transfected into Human Mesenchymal Stem Cells (hMSCs) using electroporation. Gene transfer efficiency, stemness, and myogenic gene profile were examined using flow cytometry, quantitative polymerase chain reaction, and RNA sequencing. In vivo efficacy of gene-transfected hMSCs was demonstrated through histological and gene expression analyses using a radiation-induced esophageal fibrosis animal model. We have confirmed that the gene transfer efficiency was high (∼75%). Pluripotency levels in gene-transfected MSCs were significantly decreased compared with those in the control (vector). Particularly, myogenesis-related genes such as OAS2, OAS3, and HSPA1A were overexpressed in the group transfected with three genes. At 4 weeks after injection, it was found that thickness collagen layer and esophageal muscle in MSCs transfected with all three genes were significantly reduced compared to those in the saline group. Muscularis mucosa was observed prominently in the gene combination group. Moreover, expression levels of myogenin, Myf6, calponin, and SM22α known to be specific markers of esophageal muscles tended to increase in the group transfected with three genes. Therefore, using gene-transfected MSCs has the potential as a promising therapy against radiation-induced esophageal fibrosis.

Optimization of the method of Agrobacterium-mediated in planta transformation of winter wheat genotypes

Aim. Optimization of the Agrobacterium-mediated in planta transformation of winter wheat genotypes. Methods. Genetic transformation, molecular genetic analysis; of mathematical statistics. Results. The influence of the composition of the inoculation medium and the method of application of the agrobacterium cell suspension on the frequency of seed setting and the formation of transgenic plants of new promising genotypes of winter wheat was studied. The dependence of obtaining the amount of grain and transformants on the genotypic characteristics of plants when using different inoculation conditions and transformation procedures was established. The higher efficiency of the MC-32 inoculation medium, which additionally contained MES, magnesium sulfate, and ammonium chloride, was shown compared to the MC-31 medium supplemented with sodium thiosulfate. A significant difference in the rate of seed setting was revealed when using different methods of applying A. tumefacins cell suspension. Method I proved to be the most effective, when a bacterial culture was applied to the pistil receptacle, and after the liquid in which the agrobacteria were resuspended completely dried, pollination was carried out with pollen obtained from an intact ear of the same plant. Conclusions. Selected inoculation conditions for efficient gene transfer in the pollination process for Agrobacterium-mediated in planta transformation of new winter wheat genotypes.

Advancements in the Controlled Delivery of Nanodrugs to the Central Nervous System: Challenges and Innovations.

About 1 million of the 6.8 million deaths attributed to disorders of the central nervous system (CNS) each year are caused by neurodegenerative diseases, which include Parkinson’s disease, Alzheimer’s disease, tumors, and ischemic stroke. Because of the complexity of the brain, CNS issues are a major concern. To address issues with toxicity, specificity, and delivery, several drugs are available to treat illnesses of the central nervous system (CNS). Therapeutic drugs are challenged by barriers such as the blood-brain barrier (BBB), which prevents them from reaching their intended target. Scholars have been investigating pathways for pharmaceuticals to cross the blood-brain barrier and reach their intended targets. These issues show how different cellular processes must be changed or manipulated by nanotechnology to obtain the required properties. Nanoparticles are an efficient replacement for drug delivery and other methods because of their nano size, which allows them to cross the blood-brain barrier. Effective drug transfer and enhanced CNS disease treatment and diagnosis are possible using nanotechnology. Drugs could be altered via nanoengineering to carry out tasks like transferring across the blood-brain barrier, modifying signaling pathways, focusing on certain cells, facilitating efficient gene transfer, and encouraging nerve cell regeneration and preservation.

Direct dorsal root ganglia (DRG) injection in mice for analysis of adeno-associated viral (AAV) gene transfer to peripheral somatosensory neurons

BackgroundDelivering optogenetic genes to the peripheral sensory nervous system provides an efficient approach to study and treat neurological disorders and offers the potential to reintroduce sensory feedback to prostheses users and those who have incurred other neuropathies. Adeno-associated viral (AAV) vectors are a common method of gene delivery due to efficiency of gene transfer and minimal toxicity. AAVs are capable of being designed to target specific tissues, with transduction efficacy determined through the combination of serotype and genetic promoter selection, as well as location of vector administration. The dorsal root ganglia (DRGs) are collections of cell bodies of sensory neurons which project from the periphery to the central nervous system (CNS). The anatomical make-up of DRGs make them an ideal injection location to target the somatosensory neurons in the peripheral nervous system (PNS). Comparison to existing methodsPrevious studies have detailed methods of direct DRG injection in rats and dorsal horn injection in mice, however, due to the size and anatomical differences between rats and strains of mice, there is only one other published method for AAV injection into murine DRGs for transduction of peripheral sensory neurons using a different methodology. New Method/ResultsHere, we detail the necessary materials and methods required to inject AAVs into the L3 and L4 DRGs of mice, as well as how to harvest the sciatic nerve and L3/L4 DRGs for analysis. This methodology results in optogenetic expression in both the L3/L4 DRGs and sciatic nerve and can be adapted to inject any DRG.

Development and Evaluation of a Shrimp Virus (IHHNV)-Mediated Gene Transfer and Expression System for Shrimps.

An efficient gene transfer and expression tool is lacking for shrimps and shrimp cells. To solve this, this study has developed a shrimp DNA virus-mediated gene transfer and expression system, consisting of insect Sf9 cells for viral packaging, the shrimp viral vector of pUC19-IHHNV-PH-GUS and the baculoviral vector of Bacmid or Bacmid-VP28 encoding the shrimp WSSV envelope protein VP28. The pUC19-IHHNV-PH-GUS vector was constructed by assembling the genomic DNA of shrimp infectious hypodermal and hematopoietic necrosis virus (IHHNV), which has shortened inverted terminal repeats, into a pUC19 backbone, and then an expression cassette of baculoviral polyhedron (PH) promoter-driven GUS (β-glucuronidase) reporter gene was inserted immediately downstream of IHHNV for proof-of-concept. It was found that the viral vector of pUC19-IHHNV-PH-GUS could be successfully packaged into IHHNV-like infective virions in the Sf9 cells, and the gene transfer efficiency of this system was evaluated and verified in three systems of Sf9 cells, shrimp hemolymph cells and tissues of infected shrimps, but the GUS expression could only be detected in cases where the viral vector was co-transfected or co-infected with a baculovirus of Bacmid or Bacmid-VP28 due to the Bacmid-dependence of the PH promoter. Moreover, the packaging and infection efficiencies could be significantly improved when Bacmid-VP28 was used instead of Bacmid.

Therapeutic Application and Structural Features of Adeno-Associated Virus Vector.

Adeno-associated virus (AAV) is characterized by non-pathogenicity, long-term infection, and broad tropism and is actively developed as a vector virus for gene therapy products. AAV is classified into more than 100 serotypes based on differences in the amino acid sequence of the capsid protein. Endocytosis involves the uptake of viral particles by AAV and accessory receptors during AAV infection. After entry into the cell, they are transported to the nucleus through the nuclear pore complex. AAVs mainly use proteoglycans as receptors to enter cells, but the types of sugar chains in proteoglycans that have binding ability are different. Therefore, it is necessary to properly evaluate the primary structure of receptor proteins, such as amino acid sequences and post-translational modifications, including glycosylation, and the higher-order structure of proteins, such as the folding of the entire capsid structure and the three-dimensional (3D) structure of functional domains, to ensure the efficacy and safety of biopharmaceuticals. To further enhance safety, it is necessary to further improve the efficiency of gene transfer into target cells, reduce the amount of vector administered, and prevent infection of non-target cells.

Stable transduction of the neonatal mouse liver using a hybrid rAAV/sleeping beauty transposon gene delivery system.

Conventional adeno-associated viral (AAV) vectors, while highly effective in quiescent cells such as hepatocytes in the adult liver, confer less durable transgene expression in proliferating cells owing to episome loss. Sustained therapeutic success is therefore less likely in liver disorders requiring early intervention. We have previously developed a hybrid, dual virion approach, recombinant AAV (rAAV)/piggyBac transposon system capable of achieving stable gene transfer in proliferating hepatocytes at levels many fold above conventional AAV vectors. An alternative transposon system, Sleeping Beauty, has been widely used for ex vivo gene delivery; however liver-targeted delivery using a hybrid rAAV/Sleeping Beauty approach remains relatively unexplored. We investigated the capacity of a Sleeping Beauty (SB)-based dual rAAV virion approach to achieve stable and efficient gene transfer to the newborn murine liver using transposable therapeutic cassettes encoding coagulation factor IX or ornithine transcarbamylase (OTC). At equivalent doses, rAAV/SB100X transduced hepatocytes with high efficiency, achieving stable expression into adulthood. Compared with conventional AAV, the proportion of hepatocytes transduced, and factor IX and OTC activity levels, were both markedly increased. The proportion of hepatocytes stably transduced increased 4- to 8-fold from <5%, and activity levels increased correspondingly, with markedly increased survival and stable urinary orotate levels in the OTC-deficient Spfash mouse following elimination of residual endogenous murine OTC. The present study demonstrates the first in vivo utility of a hybrid rAAV/SB100X transposon system to achieve stable long-term therapeutic gene expression following delivery to the highly proliferative newborn mouse liver. These results have relevance to the treatment of genetic metabolic liver diseases with neonatal onset.

Gene transfer of human cardiomyopathy β-MyHC mutant R403Q directly alters intact cardiac myocyte calcium homeostasis and causes hyper-contractility.

The R403Q mutation of human cardiac β-myosin heavy chain was the first missense mutation of a sarcomeric protein identified as being causal for hypertrophic cardiomyopathy (HCM), in humans. The direct effect of the R403Q mutant myosin on intracellular calcium homeostasis and contractility is not fully known. Here we have used in vitro gene transfer of the R403Q mutant human β-myosin to study its direct effects on single intact adult cardiac myocyte contractility and calcium homeostasis. In the first experiments, adult cardiac myocytes transduced with the R403Q mutant myosin recombinant viral vectors were compared to myocytes transduced with wild-type human β-myosin (wtMYH7). Efficiency of gene transfer was high in both groups (>98%) and the degree of stoichiometric myofilament incorporation of either the mutant or normal myosin was comparable at ∼40% in quiescent myocytes in primary culture. Sarcomere structure and cellular morphology were unaffected by R403Q myosin expression and myofilament incorporation. Functionally, in electrically paced cardiac myocytes, the R403Q mutant myosin caused a significant increase in intracellular calcium concentration and myocyte hyper-contractility. At the sub-cellular myofilament level, the mutant myosin increased the calcium sensitivity of steady state isometric tension development and increased isometric cross-bridge cycling kinetics. R403Q myocytes became arrhythmic after β-adrenergic stimulation with spontaneous calcium transients and contractions in between electrical stimuli. These results indicate that human R403Q mutant myosin directly alters myofilament function and intracellular calcium cycling. Elevated calcium levels may provide a trigger for the ensuing hypertrophy and susceptibility to arrhythmia that are characteristic of HCM.

Lipoplexes' Structure, Preparation, and Role in Managing Different Diseases.

Lipid-based vectors are becoming promising alternatives to traditional therapies over the last 2 decades specially for managing life-threatening diseases like cancer. Cationic lipids are the most prevalent non-viral vectors utilized in gene delivery.The increasing number of clinical trials about lipoplex-based gene therapy demonstrates their potential as well-established technology that can provide robust gene transfection. In this regard, this review will summarize this important point.These vectors however have a modest transfection efficiency. This limitation can be partly addressed by using functional lipids that provide a plethora of options for investigating nucleic acid-lipid interactions as well as in vitro and in vivo nucleic acid delivery for biomedical applications. Despite their lower gene transfer efficiency, lipid-based vectors such as lipoplexes have several advantages over viral ones: they are less toxic and immunogenic, can be targeted, and are simple to produce on a large scale. Researchers are actively investigating the parameters that are essential for an effective lipoplex delivery method. These include factors that influence the structure, stability, internalization, and transfection of the lipoplex.Thorough understanding of the design principles will enable synthesis of customized lipoplex formulations for life-saving therapy.

Adeno-associated virus (AAV) 6 provides efficient gene transfer upon intramyocardial injection in vivo

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): European Innovation Council Eurostars Background Cardiac gene transfer utilizing adeno-associated virus (AAV) is a promising approach for the treatment of various cardiovascular diseases. Intramyocardial injection is an efficient delivery route, and is particularly relevant for ischemic disease or pacemaker and conduction system abnormalities, where local treatment is favoured. However, which AAV variants provide efficient cardiac gene transfer after intramyocardial injection remains to be tested. Aim This study aims to compare several cardiotropic AAV variants in order to identify their relative potency in the setting of direct intramyocardial injection. Methods A green fluorescent protein (GFP)-coding sequence was packaged in the two benchmark AAV capsids, AAV6 and AAV9, and a selection of recently reported AAV9 capsid variants which are named MyoAAVs. These AAV vectors were added to neonatal rat ventricular cardiomyocytes (NRVM) and their transduction efficiency was evaluated by flow cytometry and quantitative PCR. AAV6, AAV9 and MyoAAV4A vectors were then delivered into the left ventricle of FVB mice via intramyocardial injection. AAV6 and AAV9 carrying GFP in a different expression cassette were also intramyocardially injected in FVB and Bl6 mice. Direct fluorescence, transgene mRNA, and viral genomes were quantified to determine the transduction efficiency 4 weeks post injection. Picrosirius red staining was used to assess whether AAV-mediated GFP delivery causes cardiac fibrosis. We further studied GFP encoding AAV6 and AAV9 vectors upon intramyocardial injection into the left ventricle of pigs and determined the transduction efficiency by quantitative PCR. Results NRVM transduction experiment revealed that among the tested capsid variants, AAV6 is the most efficient in transducing cardiomyocytes, followed by MyoAAV4A. In mice, AAV6 is most effective in transducing myocardium upon intramyocardial injection, resulting in approximately 10-fold higher transgene expression than that obtained with AAV9 vector and 2-fold higher than that with MyoAAV4A vector. Histology revealed no vector-related cardiac fibrosis. The use of different expression cassettes and animal strains consistently showed that AAV6 outperforms AAV9 in cardiac transduction efficiency. Transgene expression in liver was only observed in Bl6 mice injected with AAV9. In porcine myocardium, AAV6 led to robust transgene expression after intramyocardial injection and this expression level is higher than that of AAV9. Conclusion Among the AAV variants tested, AAV6 appears to be the most efficient for cardiac transduction by intramyocardial injection. These results suggest that AAV6 represents an attractive vector for direct cardiac gene transfer and for future capsid engineering in the context of local transgene delivery.

Development of KoRV-pseudotyped lentiviral vectors for efficient gene transfer into freshly isolated immune cells

Allogeneic cell therapies, such as those involving macrophages or Natural Killer (NK) cells, are of increasing interest for cancer immunotherapy. However, the current techniques for genetically modifying these cell types using lenti- or gamma-retroviral vectors present challenges, such as required cell pre-activation and inefficiency in transduction, which hinder the assessment of preclinical efficacy and clinical translation. In our study, we describe a novel lentiviral pseudotype based on the Koala Retrovirus (KoRV) envelope protein, which we identified based on homology to existing pseudotypes used in cell therapy. Unlike other pseudotyped viral vectors, this KoRV-based envelope demonstrates remarkable efficiency in transducing freshly isolated primary human NK cells directly from blood, as well as freshly obtained monocytes, which were differentiated to M1 macrophages as well as B cells from multiple donors, achieving up to 80% reporter gene expression within three days post-transduction. Importantly, KoRV-based transduction does not compromise the expression of crucial immune cell receptors, nor does it impair immune cell functionality, including NK cell viability, proliferation, cytotoxicity as well as phagocytosis of differentiated macrophages. Preserving immune cell functionality is pivotal for the success of cell-based therapeutics in treating various malignancies. By achieving high transduction rates of freshly isolated immune cells before expansion, our approach enables a streamlined and cost-effective automated production of off-the-shelf cell therapeutics, requiring fewer viral particles and less manufacturing steps. This breakthrough holds the potential to significantly reduce the time and resources required for producing e.g. NK cell therapeutics, expediting their availability to patients in need.

A novel high-titer, bifunctional lentiviral vector for autologous hematopoietic stem cell gene therapy of sickle cell disease

A major limitation of gene therapy for sickle cell disease (SCD) is the availability and access to a potentially curative one-time treatment, due to high treatment costs. We have developed a high-titer bifunctional lentiviral vector (LVV) in a vector backbone that has reduced size, high vector yields, and efficient gene transfer to human CD34+ hematopoietic stem and progenitor cells (HSPC). This LVV contains locus control region cores expressing an anti-sickling βAS3-globin gene and two microRNA adapted short hairpin RNA simultaneously targeting BCL11A and ZNF410 transcripts to maximally induce fetal hemoglobin (HbF) expression. This LVV induces high levels of anti-sickling hemoglobins (HbAAS3 + HbF), while concurrently reducing sickle hemoglobin (HbS). The reduction in HbS and increased anti-sickling hemoglobin impedes deoxygenated HbS polymerization and red blood cell sickling at low vector copy per cell in transduced SCD patient CD34+ cells differentiated into erythrocytes. The dual alterations in red cell hemoglobins ameliorated the SCD phenotype in the SCD Berkeley mouse model in vivo. With high titer and enhanced transduction of HSPC at low multiplicity of infection, this LVV will increase the number of patient doses of vector from production lots to reduce costs and help improve accessibility to gene therapy for SCD.

Drying-wetting cycle enhances stress resistance of Escherichia coli O157:H7 in a model soil

Outbreaks of Escherichia coli (E. coli) O157:H7 in farms are often triggered by heavy rains and flooding. Most cells die with the decreasing of soil moisture, while few cells enter a dormant state and then resuscitate after rewetting. The resistance of dormant cells to stress has been extensively studied, whereas the molecular mechanisms of the cross-resistance development of the resuscitated cells are poorly known. We performed a comparative proteomic analysis on O157:H7 before and after undergoing soil dry-wet alternation. A differential expression of 820 proteins was identified in resuscitated cells compared to exponential-phase cells, as determined by proteomics analysis. The GO and KEGG pathway enrichment analyses revealed that up-regulated proteins were associated with oxidative phosphorylation, glycolysis/gluconeogenesis, the citrate cycle (TCA cycle), aminoacyl-tRNA biosynthesis, ribosome activity, and transmembrane transporters, indicating increased energy production and protein synthesis in resuscitated O157:H7. Moreover, proteins related to acid, osmotic, heat, oxidative, antibiotic stress and horizontal gene transfer efficiency were up-regulated, suggesting a potential improvement in stress resistance. Subsequent validation experiments demonstrated that the survival rates of the resuscitated cells were 476.54 and 7786.34 times higher than the exponential-phase cells, with pH levels of 1.5 and 2.5, respectively. Similarly, resuscitated cells showed higher survival rates under osmotic stress, with 7.5%, 15%, and 30% NaCl resulting in survival rates that were 460.58, 1974.55, and 3475.31 times higher. Resuscitated cells also exhibited increased resistance to heat stress, with survival rates 69.64 and 139.72 times higher at 55 °C and 90 °C, respectively. Furthermore, the horizontal gene transfer (HGT) efficiency of resuscitated cells was significantly higher (153.12-fold) compared to exponential phase cells. This study provides new insights into bacteria behavior under changing soil moisture and this may explain O157:H7 outbreaks following rainfall and flooding, as the dry-wet cycle promotes stress cross-resistance development.

340 Anti-CD20 attenuates lung humoral and cellular immune responses: implications for gene therapy in Cystic Fibrosis

OBJECTIVES/GOALS: Re-administration of inhaled gene therapies has the potential to overcome low correction efficiencies and limiting immune responses observed in previous trials of gene therapy for Cystic Fibrosis. We therefore tested the hypothesis that pre-treatment with a B-cell depleting α ± CD20 antibody would permit vector re-administration. METHODS/STUDY POPULATION: We first selected Adenoviral (Ad) vectors to study initially due to their well-known ability to elicit potent immune responses. Mice were dosed with a depleting α ± CD20 antibody or isotype control 2 days prior to delivery of Ad-Luc. 4 weeks later, mice were euthanized to assess the development of anti-vector immune responses. Flow cytometry and single-cell RNA sequencing were used to evaluate the development of lung-resident memory cells. Serum and airway antibody responses were assessed by ELISA. After 4 weeks, mice were dosed with Ad-LacZ and euthanized 3 days later to assess efficiency of second-round gene transfer by β-galactosidase activity assay. Similar methods were used in a pilot experiment with Adeno-associated virus vector (AAV), but with euthanasia 3 weeks after secondary gene transfer. RESULTS/ANTICIPATED RESULTS: Delivery of Ad vectors leads to the development of lung-resident memory B and T-cells. The depletion of B-cells prior to first-round vector delivery attenuated airway T-cell infiltration and serum IgG production, abrogated mucosal IgG and IgA production, and completely rescued secondary gene transfer. Genetically modified mouse models suggest secreted antibodies are critical in prevention of vector redosing. AAV vectors were found to be less immunogenic than Ad vectors, with only partial reduction of second-round gene transfer. However, anti-CD20 provided no benefit for AAV redelivery. DISCUSSION/SIGNIFICANCE: Mucosal humoral immunity is critical in preventing re-administration of Adenoviral vectors. Impairment of B-cell responses by α ± CD20 treatment prior to vector delivery allows re-administration and may help overcome low efficiencies of CF gene therapy. AAV vectors may be less susceptible to neutralization by pre-existing immunity.

Fine-tuning FAM161A gene augmentation therapy to restore retinal function

For 15 years, gene therapy has been viewed as a beacon of hope for inherited retinal diseases. Many preclinical investigations have centered around vectors with maximal gene expression capabilities, yet despite efficient gene transfer, minimal physiological improvements have been observed in various ciliopathies. Retinitis pigmentosa-type 28 (RP28) is the consequence of bi-allelic null mutations in the FAM161A, an essential protein for the structure of the photoreceptor connecting cilium (CC). In its absence, cilia become disorganized, leading to outer segment collapses and vision impairment. Within the human retina, FAM161A has two isoforms: the long one with exon 4, and the short one without it. To restore CC in Fam161a-deficient mice shortly after the onset of cilium disorganization, we compared AAV vectors with varying promoter activities, doses, and human isoforms. While all vectors improved cell survival, only the combination of both isoforms using the weak FCBR1-F0.4 promoter enabled precise FAM161A expression in the CC and enhanced retinal function. Our investigation into FAM161A gene replacement for RP28 emphasizes the importance of precise therapeutic gene regulation, appropriate vector dosing, and delivery of both isoforms. This precision is pivotal for secure gene therapy involving structural proteins like FAM161A.

A humanized mouse model for adeno-associated viral gene therapy

Clinical translation of AAV-mediated gene therapy requires preclinical development across different experimental models, often confounded by variable transduction efficiency. Here, we describe a human liver chimeric transgene-free Il2rg−/−/Rag2−/−/Fah−/−/Aavr−/− (TIRFA) mouse model overcoming this translational roadblock, by combining liver humanization with AAV receptor (AAVR) ablation, rendering murine cells impermissive to AAV transduction. Using human liver chimeric TIRFA mice, we demonstrate increased transduction of clinically used AAV serotypes in primary human hepatocytes compared to humanized mice with wild-type AAVR. Further, we demonstrate AAV transduction in human teratoma-derived primary cells and liver cancer tissue, displaying the versatility of the humanized TIRFA mouse. From a mechanistic perspective, our results support the notion that AAVR functions as both an entry receptor and an intracellular receptor essential for transduction. The TIRFA mouse should allow prediction of AAV gene transfer efficiency and the study of AAV vector biology in a preclinical human setting.

Optimization strategies and advances in the research and development of AAV-based gene therapy to deliver large transgenes.

Adeno-associated virus (AAV)-based therapies are recognized as one of the most potent next-generation treatments for inherited and genetic diseases. However, several biological and technological aspects of AAV vectors remain a critical issue for their widespread clinical application. Among them, the limited capacity of the AAV genome significantly hinders the development of AAV-based gene therapy. In this context, genetically modified transgenes compatible with AAV are opening up new opportunities for unlimited gene therapies for many genetic disorders. Recent advances in de novo protein design and remodelling are paving the way for new, more efficient and targeted gene therapeutics. Using computational and genetic tools, AAV expression cassette and transgenic DNA can be split, miniaturized, shuffled or created from scratch to mediate efficient gene transfer into targeted cells. In this review, we highlight recent advances in AAV-based gene therapy with a focus on its use in translational research. We summarize recent research and development in gene therapy, with an emphasis on large transgenes (>4.8kb) and optimizing strategies applied by biomedical companies in the research pipeline. We critically discuss the prospects for AAV-based treatment and some emerging challenges. We anticipate that the continued development of novel computational tools will lead to rapid advances in basic gene therapy research and translational studies.

Advances of Recombinant Adenoviral Vectors in Preclinical and Clinical Applications.

Adenoviruses (Ad) have the potential to induce severe infections in vulnerable patient groups. Therefore, understanding Ad biology and antiviral processes is important to comprehend the signaling cascades during an infection and to initiate appropriate diagnostic and therapeutic interventions. In addition, Ad vector-based vaccines have revealed significant potential in generating robust immune protection and recombinant Ad vectors facilitate efficient gene transfer to treat genetic diseases and are used as oncolytic viruses to treat cancer. Continuous improvements in gene delivery capacity, coupled with advancements in production methods, have enabled widespread application in cancer therapy, vaccine development, and gene therapy on a large scale. This review provides a comprehensive overview of the virus biology, and several aspects of recombinant Ad vectors, as well as the development of Ad vector, are discussed. Moreover, we focus on those Ads that were used in preclinical and clinical applications including regenerative medicine, vaccine development, genome engineering, treatment of genetic diseases, and virotherapy in tumor treatment.