Levels Of Transgene Expression Research Articles

Entrapment of lipid nanoparticles in peripheral endosomes but not lysosomes impairs intracellular trafficking and endosomal escape

The uptake and intracellular trafficking of lipid nanoparticles (LNPs) along the endolysosomal pathway leading to releasing compartments is critical for delivery efficiency. How the players of the processes interact with each other to affect LNP delivery remains unclear. Here, we employed a recently developed, highly sensitive LNP labeling platform in combination with known-state of endolysosomal activity of cells to address this outstanding question with spatiotemporal analysis. We found the endolysosomal activity (endolyosomal pH, endolysosomal protease activation), which was regulated by nutrients, determines the active endocytosis activity of cells. Elevated internalization of DNA and LNP alike by cells correlated with increased endolysosomal activity. Similar to naked DNA, elevated internalization of LNP resulted in entrapment of LNPs in peripheral endosomes, which significantly impaired the intracellular trafficking of LNP to the perinuclear lysosome region and cytosolic release of LNP cargo. On the other hand, we found the extent of perinuclear lysosomal LNP accumulation positively correlated with the level of transgene expression. Moreover, we found continuous internalization of LNP was necessary not only to saturate the degradation compartments to overcome rapid degradation of LNP but also to maintain a necessary pool of releasing compartments, shuttling between peripheral endosomes and lysosomes via anterograde transport and retrograde transport along microtubules respectively, for meaningful endosomal release. Our results suggest the balance between endocytosis and intracellular trafficking needs to be fine-tuned according to endolysosomal activity of target cell to achieve optimal cytosol release.

Cyclosporin H Improves the Transduction of CD34+ Cells with an Anti-Sickling Globin Vector, a Possible Therapeutic Approach for Sickle Cell Disease.

Sickle cell disease (SCD) is a debilitating monogenic disease originating from mutations in the hemoglobin beta chain gene producing an abnormal hemoglobin HbS. The polymerization of HbS is responsible for the sickling of erythrocytes leading to anemia and vaso-occlusive events. Gene therapy is a promising treatment of SCD, and two different gene therapy drugs, using gene editing or gene transfer, have already reached the marketing stage. There is still a need to improve the efficacy of gene therapy in SCD, particularly when using anti-sickling beta-globin gene transfer strategies, which must outcompete the pathological HbS. One possibility is to increase transduction by inhibiting lentiviral restriction factors such as interferon-induced transmembrane proteins (IFITMs). This can be achieved by the addition of cyclosporin H (CsH) during the transduction process. This strategy was applied here in CD34+ hematopoietic progenitor and stem cells obtained from cord blood (CB). A first series of experiments with lentiviral vector coding for a green fluorescent protein (GFP) gene confirmed that the addition of CsH enhanced transgene expression levels and vector copy number per cell (VCN), while CD34+ cells remained viable and functional. Notably, the production of colony-forming cells (CFC) remained unaffected unless very high VCN values were reached. In a second step, CD34+ cells obtained from the CB of newborns with homozygous (n = 2) or heterozygous (n = 1) SCD mutations were transduced with the GLOBE-AS3 lentiviral vector coding for the HbAS3 anti-sickling beta globin. As with GFP, GLOBE-AS3 lentiviral transduction was clearly enhanced by CsH, leading to VCN > 2 and therapeutic levels of expression of the HbAS3. Moreover, the process did not affect the viability or functions of CFC. The combination of CB progenitors, the GLOBE-AS3 vector, and CsH is thus shown here to be a promising approach for the treatment of SCD.

Features of the eGFP Gene Expression in Transplastomic Tobacco Plants

Transplastomic tobacco plants expressing a reporter gene egfp and selectable marker gene aadA as part of a bicistronic operon were obtained using the bioballistics method. Features of egfp gene expression were studied in two groups of transplastomic plants: seed progeny obtained from self-pollination and plants obtained as a result of two successive regeneration cycles from the leaves of the original transformants. A comparative fluorimetric analysis of the recombinant protein accumulation in groups of transplastomic plants and nuclear transformants was carried out. It was found that the amount of recombinant eGFP protein accumulated in the leaves of transplastomic plants was unexpectedly low and did not exceed the level established for nuclear transformants. Real-time PCR results showed that the low level of recombinant eGFP accumulation was not associated with low levels of transgene expression or the presence of nontransgenic copies of the plastid genome in chloroplasts. This is most likely due to limitations imposed at the level of translation of recombinant proteins in chloroplasts.

Posttranscriptional tuning of gene expression over a large dynamic range in synthetic tobacco chloroplast operons.

Achieving optimally balanced gene expression within synthetic operons requires regulatory elements capable of providing a spectrum of expression levels. In this study, we investigate the expression of gfp reporter gene in tobacco chloroplasts, guided by variants of the plastid atpH 5' UTR, which harbors a binding site for PPR10, a protein that activates atpH at the posttranscriptional level. Our findings reveal that endogenous tobacco PPR10 confers distinct levels of reporter activation when coupled with the tobacco and maize atpH 5' UTRs in different design contexts. Notably, high GFP expression was not coupled to the stabilization of monocistronic gfp transcripts in dicistronic reporter lines, adding to the evidence that PPR10 activates translation via a mechanism that is independent of its stabilization of monocistronic transcripts. Furthermore, the incorporation of a tRNA upstream of the UTR nearly abolishes gfp mRNA (and GFP protein), presumably by promoting such rapid RNA cleavage and 5' exonucleolytic degradation that PPR10 had insufficient time to bind and protect gfp RNA, resulting in a substantial reduction in GFP accumulation. When combined with a mutant atpH 5' UTR, the tRNA leads to an exceptionally low level of transgene expression. Collectively, this approach allows for tuning of reporter gene expression across a wide range, spanning from a mere 0.02-25% of the total soluble cellular protein. These findings highlight the potential of employing cis-elements from heterologous species and expand the toolbox available for plastid synthetic biology applications requiring multigene expression at varying levels.

An optimal promoter regulating cytokine transgene expression is crucial for safe and effective oncolytic virus immunotherapy

In general, ensuring safety is the top priority of a new modality. Although oncolytic virus armed with an immune stimulatory transgene (OVI) showed some promise, the strategic concept of simultaneously achieving maximum effectiveness and minimizing side effects has not been fully explored. We generated a variety of survivin-responsive “conditionally replicating adenoviruses that can target and treat cancer cells with multiple factors (m-CRAs)” (Surv.m-CRAs) armed with the granulocyte-macrophage colony-stimulating factor (GM-CSF) transgene downstream of various promoters using our m-CRA platform technology. We carefully analyzed both therapeutic and adverse effects of them in the in vivo syngeneic Syrian hamster cancer models. Surprisingly, an intratumor injection of a conventional OVI, which expresses the GM-CSF gene under the constitutively and strongly active “cytomegalovirus enhancer and β-actin promoter”, provoked systemic and lethal GM-CSF circulation and shortened overall survival (OS). In contrast, a new conceptual type of OVI, which expressed GM-CSF under the cancer-predominant and mildly active E2F promoter or the moderately active “Rous sarcoma virus long terminal repeat”, not only abolished lethal adverse events but also prolonged OS and systemic anti-cancer immunity. Our study revealed a novel concept that optimal expression levels of an immune stimulatory transgene regulated by a suitable upstream promoter is crucial for achieving high safety and maximal therapeutic effects simultaneously in OVI therapy. These results pave the way for successful development of the next-generation OVI and alert researchers about possible problems with ongoing clinical trials.

Peripheral Cellular Immune Responses Induced by Subretinal Adeno-Associated Virus Gene Transfer Can Be Restrained by the Subretinal-Associated Immune Inhibition Mechanism.

After more than two decades of basic research and preclinical studies, adeno-associated virus (AAV)-mediated gene transfer has been tested successfully in clinical trials to treat inherited retinal diseases. Despite the eye's immune-privileged status, some patients display inflammatory events requiring the use of corticoids as an adjunct treatment which led us to question the immune consequences of a subretinal AAV administration. We first characterized anti-transgene immune responses induced in the periphery by injecting increasing doses of AAV8 encoding reporter proteins fused with the HY male antigen into the subretinal space of female C57BL/6 and rd10 mice. Transgene expression was monitored over time with bioluminescence imaging, and T cell immune responses in the spleen were analyzed by IFNγ ELISpot and cytokine multiplex assays. Our data show that AAV8 injections cause pro-inflammatory T cell immune response against the transgene product correlated with the transgene expression level at 2.109 vg and above. In addition, co-injection of immunodominant peptides from the transgene product, along with AAV8, modulates the immune response at all AAV doses tested. Taken together, our data suggest that injection of AAV8 in the subretinal space induces pro-inflammatory peripheral T cell responses to the transgene product that can be modulated by the subretinal-associated immune inhibition mechanism.

Gene Therapy for Hemophilia B: Achievements, Open Issues, and Perspectives.

Hemophilia B is the first bleeding disorder for which gene therapy clinical programs began. Presently, adenovirus-associated vectors represent the best means to deliver the transgene, and their administration by intravenous route has been used in recent clinical trials. The natural occurring factor IX (FIX) Padua variant, which allows for a 5- to 8-fold higher activity of FIX, while maintaining a normal protein concentration, was subsequently used to enhance the level of transgene expression. All the recent trials using this variant showed good results, and accumulating data suggest that long-term expression durability could be maintained at a significant hemostatic level. However, the risk of loss of transgene expression associated to immune response with liver enzymes elevation remains a concern, especially as to the efficacy and duration of immunosuppressive treatment. Notwithstanding this limitation, the results of clinical trials suggest that gene therapy in hemophilia B has the potential to provide long-term benefits with sustained factor activity levels predicted to last several years in many patients.

OCT Intensity of the Region between Outer Retina Band 2 and Band 3 as a Biomarker for Retinal Degeneration and Therapy.

Optical coherence tomography (OCT) is widely used to probe retinal structure and function. This study investigated the outer retina band (ORB) pattern and reflective intensity for the region between bands 2 and 3 (Dip) in three mouse models of inherited retinal degeneration (Rs1KO, TTLL5KO, RPE65KO) and in human AMD patients from the A2A database. OCT images were manually graded, and reflectivity signals were used to calculate the Dip ratio. Qualitative analyses demonstrated the progressive merging band 2 and band 3 in all three mouse models, leading to a reduction in the Dip ratio compared to wildtype (WT) controls. Gene replacement therapy in Rs1KO mice reverted the ORB pattern to one resembling WT and increased the Dip ratio. The degree of anatomical rescue in these mice was highly correlated with level of transgenic RS1 expression and with the restoration of ERG b-wave amplitudes. While the inner retinal cavity was significantly enlarged in dark-adapted Rs1KO mice, the Dip ratio was not altered. A reduction of the Dip ratio was also detected in AMD patients compared with healthy controls and was also positively correlated with AMD severity on the AMD score. We propose that the ORB and Dip ratio can be used as non-invasive early biomarkers for retina health, which can be used to probe therapeutic gene expression and to evaluate the effectiveness of therapy.

Oschib1 gene encoding a GH18 chitinase confers resistance against sheath blight disease of rice caused by Rhizoctonia solani AG1-IA.

Sheath blight disease of rice caused by Rhizoctonia solani AG1-IA, is a major fungal disease responsible for huge loss to grain yield and quality. The major limitation of achieving persistent and reliable resistance against R. solani is the governance of disease resistance trait by many genes. Therefore, functional characterization of new genes involved in sheath blight resistance is necessary to understand the mechanism of resistance as well as evolving effective strategies to manage the disease through host-plant resistance. In this study, we performed RNA sequencing of six diverse rice genotypes (TN1, BPT5204, Vandana, N22, Tetep, and Pankaj) from sheath and leaf tissue of control and fungal infected samples. The approach for identification of candidate resistant genes led to identification of 352 differentially expressed genes commonly present in all the six genotypes. 23 genes were analyzed for RT-qPCR expression which helped identification of Oschib1 showing differences in expression level in a time-course manner between susceptible and resistant genotypes. The Oschib1 encoding classIII chitinase was cloned from resistant variety Tetep and over-expressed in susceptible variety Taipei 309. The over-expression lines showed resistance against R. solani, as analyzed by detached leaf and whole plant assays. Interestingly, the resistance response was correlated with the level of transgene expression suggesting that the enzyme functions in a dose dependent manner. We report here the classIIIb chitinase from chromosome10 of rice showing anti-R. solani activity to combat the dreaded sheath blight disease.

Overexpression of Slc22a18 facilitates fat accumulation in mice

We previously reported that solute carrier family 22 member 18 (Slc22a18) regulates lipid accumulation in 3T3-L1 adipocytes. Here, we provide additional evidence derived from experiments with adenoviral vector expression and genetic manipulation of mice. In primary cultured rat hepatocytes, adenoviral overexpression of mouse Slc22a18 increased triglyceride accumulation and triglyceride synthetic activity, which was decreased in an adenoviral knockdown experiment. Adenoviral overexpression of mouse Slc22a18 in vivo caused massive fatty liver in mice, even under normal dietary conditions. Conversely, adenoviral knockdown of mouse Slc22a18 reduced hepatic lipid accumulation induced by a high-glucose and high-sucrose diet. We created Slc22a18 knockout mice, which grew normally and showed no obvious spontaneous phenotypes. However, compared with control littermates, the knockout mice exhibited decreased hepatic triglyceride content under refeeding conditions, significantly reduced epididymal fat mass, and tended to have lower liver weight in conjunction with leptin deficiency. Finally, we created transgenic mice overexpressing rat Slc22a18 in an adipose-specific manner, which had increased body weight and epididymal fat mass primarily because of increased adipocyte cell volume. In these transgenic mice, a positive correlation was observed between adiposity and the expression levels of the rat Slc22a18 transgene. Taken together, these results indicate that Slc22a18 has positive effects on lipid accumulation in vivo.

Abstract 7248: Simultaneous isolation and parallel analysis of genomic DNA and RNA for gene therapy

Abstract Introduction Viral vectors, such as adeno-associated vector (AAV), play a crucial role in delivering transgenes for gene therapy research. The success of vector transduction is vital for efficacy, which is measured through vector copy number (VCN) and transgene expression. In preclinical studies, both VCN and transgene expression are commonly tested from the same sample to ensure accuracy. We have reported a method for simultaneous high-quality genomic DNA and RNA extraction without splitting the sample, utilizing SPRI bead-based technology that has consistent performance across diverse tissue types and brain regions. Methods AAV was administered to mouse and non-human primate (NHP) models. At the conclusion of each experiment, tissue samples from the adrenal gland, brain, gonads, heart, kidney, liver, lung, skeletal muscle, spinal cord, and spleen were harvested. All tissue samples that demonstrated around 15 mm3 were snap-frozen in liquid nitrogen before nucleic acid extraction. The extraction process was carried out manually or on an automated liquid handler. The quality and quantity of DNA and RNA were then assessed from all tested tissues before downstream assay. Results Our study found that all mouse tissue types (n=72/tissue type) and NHP (n=11/tissue type) had an average gDNA and RNA yield that exceeded the minimum yield cutoff of 25 ng/μL for DNA and 6.25 ng/μL for RNA. We also analyzed DNA from tissues treated with two different AAV serotype-treated tissues in the mouse model, which showed that transduction across all the tissues was highly efficient for both AAV serotypes. Furthermore, we observed variations in transgene expression levels (copies/1 μg RNA) among different tissue types. Similar patterns of VCN and transgene expression were also observed in NHP models. Conclusions Efficient gene therapy depends on a safe and effective viral vector. The simultaneous extraction of gDNA and RNA simplifies the process, saves time, ensures quality, and facilitates integration into existing lab automation. This leads to consistent and error-resistant performance in analyzing vector copy and transgene levels. Citation Format: Han Wei. Simultaneous isolation and parallel analysis of genomic DNA and RNA for gene therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7248.

Delivery of a Hepatitis C Virus Vaccine Encoding NS3 Linked to the MHC Class II Chaperone Protein Invariant Chain Using Bacterial Ghosts.

Efficient delivery of a DNA plasmid into antigen-presenting cells (APCs) is a potential strategy to enhance the immune responses of DNA vaccines. The bacterial ghost (BG) is a potent DNA vaccine delivery system that targets APCs. In the present work, we describe a new strategy of using E. coli BGs as carriers for an Ii-linked Hepatitis C Virus (HCV) NS3 DNA vaccine that improved both the transgene expression level and the antigen-presentation level in APCs. BGs were prepared from DH5α cells, characterized via electron microscopy and loaded with the DNA vaccine. The high transfection efficiency mediated using BGs was first evaluated in vitro, and then, the immune protective effect of the BG-Ii-NS3 vaccine was determined in vivo. It was found that the antibody titer in the sera of BG-Ii-NS3-challenged mice was higher than that of Ii-NS3-treated mice, indicating that the BGs enhanced the humoral immune activity of Ii-NS3. The cellular immune protective effect of the BG-Ii-NS3 vaccine was determined using long-term HCV NS3 expression in a mouse model in which luciferase was used as a reporter for HCV NS3 expression. Our results showed that the luciferase activity in BG-Ii-NS3-treated mice was significantly reduced compared with that in Ii-NS3-treated mice. The CTL assay results demonstrated that BG-Ii-NS3 induced a greater NS3-specific T-cell response than did Ii-NS3. In summary, our study demonstrated that BGs enhanced both the humoral and cellular immune response to the Ii-NS3 DNA vaccine and improved its immune protection against HCV infection.

B cell focused transient immune suppression protocol for efficient AAV readministration to the liver

Adeno associated virus (AAV) vectors are utilized for correcting multiple genetic disorders. Although the goal is to achieve life-long correction with a single vector administration, the ability to redose would enable extension of therapy in cases where initial gene transfer is insufficient to achieve a lasting cure, episomal vector forms are lost in growing organs of pediatric patients, or transgene expression is diminished over time. However, AAV typically induces potent and long-lasting neutralizing antibodies (NAb) against capsid that prevents re-administration. To prevent NAb formation in hepatic AAV8 gene transfer, we developed a transient B cell targeting protocol using a combination of monoclonal antibody therapy against CD20 (for B cell depletion) and BAFF (to slow B cell repopulation). Initiation of immunosuppression prior to (rather than at the time of) vector administration and prolonged anti-BAFF treatment prevented immune responses against the transgene product and abrogated prolonged IgM formation. As a result, vector re-administration after immune reconstitution was highly effective. Interestingly, re-administration before the immune system had fully recovered achieved further elevated levels of transgene expression. Finally, this immunosuppression protocol reduced Ig mediated AAV uptake by immune cell types with implications to reduce the risk of immunotoxicities in human gene therapy with AAV.

Cloning of maize chitinase 1 gene and its expression in genetically transformed rice to confer resistance against rice blast caused by Pyricularia oryzae.

Fungal pathogens are one of the major reasons for biotic stress on rice (Oryza sativa L.), causing severe productivity losses every year. Breeding for host resistance is a mainstay of rice disease management, but conventional development of commercial resistant varieties is often slow. In contrast, the development of disease resistance by targeted genome manipulation has the potential to deliver resistant varieties more rapidly. The present study reports the first cloning of a synthetic maize chitinase 1 gene and its insertion in rice cv. (Basmati 385) via Agrobacterium-mediated transformation to confer resistance to the rice blast pathogen, Pyricularia oryzae. Several factors for transformation were optimized; we found that 4-week-old calli and an infection time of 15 minutes with Agrobacterium before colonization on co-cultivation media were the best-suited conditions. Moreover, 300 μM of acetosyringone in co-cultivation media for two days was exceptional in achieving the highest callus transformation frequency. Transgenic lines were analyzed using molecular and functional techniques. Successful integration of the gene into rice lines was confirmed by polymerase chain reaction with primer sets specific to chitinase and hpt genes. Furthermore, real-time PCR analysis of transformants indicated a strong association between transgene expression and elevated levels of resistance to rice blast. Functional validation of the integrated gene was performed by a detached leaf bioassay, which validated the efficacy of chitinase-mediated resistance in all transgenic Basmati 385 plants with variable levels of enhanced resistance against the P. oryzae. We concluded that overexpression of the maize chitinase 1 gene in Basmati 385 improved resistance against the pathogen. These findings will add new options to resistant germplasm resources for disease resistance breeding. The maize chitinase 1 gene demonstrated potential for genetic improvement of rice varieties against biotic stresses in future transformation programs.

Obtaining of transgenic barrelclover plants (Medicago truncatula) producing chicken interferon gamma for veterinary use

At the Laboratory of Plant Genetic and Cellular Engineering, Department of Genetics and Biotechnology, St. Petersburg State University, five transgenic Medicago truncatula plants were obtained through Agrobacterium-mediated transformation, carrying one of the variants of the heterologous chicken interferon-gamma gene under the control of the constitutive 35S CaMV promoter. Among these, one plant harbored an unmodified gene insertion, while four had a modified gene with a deletion at the protease recognition site, providing resistance to proteolytic degradation.
 We demonstrate the application of the SWPOP-PCR “genome walking” method to determine the integration sites of T-DNA into the plant genome, identify the number of insertion copies and their orientation. Analysis of the obtained sequences revealed that only one plant exhibited a single T-DNA insertion, which represents the most optimal structure for stable expression.
 Upon self-pollination of T0 plants, 39 offspring were obtained and subjected to testing for the presence and expression of the transgene. Among them, six homozygous plants were identified using molecular methods. Quantitative assessment of transgene expression levels showed significant differences among representatives of different lines and among the offspring derived from a single transformed plant. Among the T1 and T2 progeny, the presence of the heterologous interferon protein in plant tissues was confirmed through Western blot analysis.
 The engineered barrelclover plants hold potential as bioreactors for the production of chicken interferon-gamma for veterinary applications. The use of an edible plant allows eliminating protein extraction and purification procedures, thereby resulting in a noteworthy reduction in production expenses of up to 80%.

Successful reversal of transgene silencing in Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii has been successfully engineered to produce compounds of interest following transgene integration and heterologous protein expression. The advantages of this model include the availability of validated tools for bioengineering, its photosynthetic ability, and its potential use as biofuel. Despite this, breakthroughs have been hindered by its ability to silence transgene expression through epigenetic changes. Histone deacetylases (HDAC) are main players in gene expression. We hypothesized that transgene silencing can be reverted with chemical treatments using HDAC inhibitors. To analyze this, we transformed C. reinhardtii, integrating into its genome the mVenus reporter gene under the HSP70-rbcs2 promoter. From 384 transformed clones, 88 (22.9%) displayed mVenus positive (mVenus+ ) cells upon flow-cytometry analysis. Five clones with different fluorescence intensities were selected. The number of integrated copies was measured by qPCR. Transgene expression levels were followed over the growth cycle and upon SAHA treatment, using a microplate reader, flow cytometry, RT-qPCR, and western blot analysis. First, we observed that expression varies with the cell cycle, reaching a maximum level just before the stationary phase in all clones. Second, we uncovered that supplementation with HDAC inhibitors of the hydroxamate family, such as vorinostat (suberoylanilide-hydroxamic-acid, SAHA) at the initiation of culture increases the frequency (% of mVenus+ cells) and the level of transgene expression per cell over the whole growth cycle, through histone deacetylase inhibition. Thus, we propose a new tool to successfully trigger the expression of heterologous proteins in the green algae C. reinhardtii, overcoming its main obstacle as an expression platform.

Integrase Defective Lentiviral Vector Promoter Impacts Transgene Expression in Target Cells and Magnitude of Vector-Induced Immune Responses.

Integrase defective lentiviral vectors (IDLVs) are a promising vaccine delivery platform given their ability to induce high magnitude and durable antigen-specific immune responses. IDLVs based on the simian immunodeficiency virus (SIV) are significantly more efficient at transducing human and simian dendritic cells (DCs) compared to HIV-based vectors, resulting in a higher expansion of antigen-specific CD8+ T cells. Additionally, IDLV persistence and continuous antigen expression in muscle cells at the injection site contributes to the durability of the vaccine-induced immune responses. Here, to further optimize transgene expression levels in both DCs and muscle cells, we generated ten novel lentiviral vectors (LVs) expressing green fluorescent protein (GFP) under different hybrid promoters. Our data show that three of the tested hybrid promoters resulted in the highest transgene expression levels in mouse DCs, monkey DCs and monkey muscle cells. We then used the three LVs with the highest in vitro transgene expression levels to immunize BALB/c mice and observed high magnitude T cell responses at 3 months post-prime. Our study demonstrates that the choice of the vector promoter influences antigen expression levels in target cells and the ensuing magnitude of T cell responses in vivo.

Engineered Human Adenoviruses of Species B and C Report Early, Intermediate Early, and Late Viral Gene Expression.

Adenoviruses (AdVs) are being developed for oncolytic or vaccination therapy against existing and emerging conditions. Well-characterized replication-competent human and human primate AdVs expressing multiple payloads are desirable, but their replication in rodent models is limited. To score the timing of adenoviral gene expression in cell cultures, we developed fully replication-competent transcriptional reporter viruses for HAdV-C5, -B3, and -B35. The picornavirus-derived 2A sequence, which induces cotranslational peptide splitting and reinitiation (skipping), was linked to GFP and the fused sequence was inserted C-terminal of the early gene E1A, the intermediate early gene protein IX and the late fiber gene. The 2A peptide induced ribosomal skipping during translation of the messenger RNA (mRNA) and gave rise to GFP from the corresponding viral promoters, as shown by immunoblotting and flow cytometry analyses of human and rodent cells. In human cells, both species B and C AdV exhibited highest reporter expression for fiber, followed by protein IX and lowest for E1A. Inoculation with either HAdV-C5 or -B3/35 viruses encoding protein IX- or fiber-GFP gave rise to higher GFP levels in hamster than mouse cells. Remarkably, despite rather low 2A ribosomal skipping efficiency of ∼50% for E1A-2A-GFP, protein IX-2A-GFP, and fiber-2A-GFP, unprocessed protein IX-2A-GFP and fiber-2A-GFP fusion proteins were efficiently incorporated into HAdV-B3 virions, respectively. These data indicate that the B3 C-termini of protein IX and fiber can be considered for retargeting engineered oncolytic or vaccination vectors, or for antigen display. The variable expression levels of transgenes from different subviral promoters may be used to improve oncolytic AdV vectors expressing therapeutic genes.

Improved efficacy of FKRP AAV gene therapy by combination with ribitol treatment for LGMD2I

Mutations in the fukutin-related protein (FKRP) gene cause dystroglycanopathy, with disease severity ranging from mild LGMD2I to severe congenital muscular dystrophy. Recently, considerable progress has been made in developing experimental therapies, with adeno-associated virus (AAV) gene therapy and ribitol treatment demonstrating significant therapeutic effect. However, each treatment has its strengths and weaknesses. AAV gene therapy can achieve normal levels of transgene expression, but it requires high doses, with toxicity concerns and variable distribution. Ribitol relies on residual FKRP function and restores limited levels of matriglycan. We hypothesized that these two treatments can work synergistically to offer an optimized therapy with efficacy and safety unmatched by each treatment alone. The most effective treatment is the combination of high-dose (5e-13 vg/kg) AAV-FKRP with ribitol, whereas low dose (1e-13 vg/kg) AAV-FKRP combined with ribitol showed a 22.6% increase in positive matriglycan fibers and the greater improvement in pathology when compared to low-dose AAV-FKRP alone. Together, our results support the potential benefits of combining ribitol with AAV gene therapy for treating FKRP-related muscular dystrophy. The fact that ribitol is a metabolite in nature and has already been tested in animal models and clinical trials in humans without severe side effects provides a safety profile for it to be trialed in combination with AAV gene therapy.

Readministration of high-dose adeno-associated virus gene therapy vectors enabled by ImmTOR nanoparticles combined with B cell-targeted agents.

Tolerogenic ImmTOR nanoparticles encapsulating rapamycin have been demonstrated to mitigate immunogenicity of adeno-associated virus (AAV) gene therapy vectors, enhance levels of transgene expression, and enable redosing of AAV at moderate vector doses of 2 to 5E12 vg/kg. However, recent clinical trials have often pushed AAV vector doses 10-fold to 50-fold higher, with serious adverse events observed at the upper range. Here, we assessed combination therapy of ImmTOR with B cell-targeting drugs for the ability to increase the efficiency of redosing at high vector doses. The combination of ImmTOR with a monoclonal antibody against B cell activation factor (aBAFF) exhibited strong synergy leading to more than a 5-fold to 10-fold reduction of splenic mature B cells and plasmablasts while increasing the fraction of pre-/pro-B cells. In addition, this combination dramatically reduced anti-AAV IgM and IgG antibodies, thus enabling four successive AAV administrations at doses up to 5E12 vg/kg and at least two AAV doses at 5E13 vg/kg, with the transgene expression level in the latter case being equal to that observed in control animals receiving a single vector dose of 1E14 vg/kg. Similar synergistic effects were seen with a combination of ImmTOR and a Bruton's tyrosine kinase inhibitor, ibrutinib. These results suggest that ImmTOR could be combined with B cell-targeting agents to enable repeated vector administrations as a potential strategy to avoid toxicities associated with vector doses above 1E14 vg/kg.