Helper-dependent Adenoviral Vectors Research Articles

Introducing a G-Makassar variant in HSCs by in vivo base editing treats sickle cell disease in mice

Precise repair of the pathogenic mutation in hematopoietic stem cells (HSCs) represents an ideal cure for patients with sickle cell disease (SCD). Here, we demonstrated correction of the SCD phenotype by converting the sickle mutation codon (GTG) into a benign G-Makassar variant (GCG) using in vivo base editing in HSCs. We demonstrated successful production of helper-dependent adenoviral vectors expressing an all-in-one base editor mapping to the sickle mutation site. In HSC-enriched cells from SCD patients, transduction with the base editing vector in vitro resulted in 35% GTG > GCG conversion and phenotypic improvements of derived red blood cells. After ex vivo transduction of HSCs from a SCD mouse model and subsequent transplantation, we achieved an average of 88% editing at the target site in transplanted mice. Importantly, in vivo HSC base editing followed by selection generated 24.5% Makassar variant in long-term repopulating HSCs of SCD mice. The treated animals demonstrated correction of disease hallmarks without showing noticeable side effects. Off-target analyses at top-scored genomic sites revealed no off-target editing. This in vivo approach requires only one non-integrating vector, only intravenous/subcutaneous injections, and minimal in vivo selection. This technically simple approach has the potential for scalable applications in resource-limiting regions where SCD is prevalent.

Abstract 3079: Overexpression Of Abca1 In Carotid Endothelium Of Hyperlipidemic Rabbits Decreases Vascular Inflammation

Introduction: Vascular gene therapy that reduces inflammation or increases cholesterol efflux might reduce atherosclerosis. ATP-binding cassette subfamily A member 1 (ABCA1) has both anti-inflammatory and cholesterol efflux activities. We tested whether vessel wall overexpression of ABCA1 would reduce inflammation, vessel wall lipid accumulation, and atherosclerotic lesion size. Methods: We used a helper-dependent adenoviral vector (HDAdABCA1) to overexpress ABCA1 in carotid endothelium of rabbits fed a normal or high-fat diet (HFD). Control arteries received a “Null” vector. We measured ABCA1 expression in transduced carotids of rabbits fed both diets. We used HFD-fed rabbits (n=30) to test whether HDAdABCA1 infusion reduced atherosclerosis. Measurements were made on whole carotid extracts, on extracts of laser-microdissected endothelium, and on histologic sections. Results: At 3 days after infusion, HDAdABCA1 increased whole vessel ABCA1 mRNA by ~2-fold in rabbits fed a normal diet (P=0.07) or a HFD (P=0.04). Endothelial ABCA1 protein was ~1.6-fold higher in HFD-fed rabbits (P=0.06). 24 wks after vector infusion in HFD-fed rabbits, endothelial ABCA1 mRNA was increased (46%; P=0.07), but whole vessel ABCA1 mRNA was slightly decreased (10%; P=0.004). Whole vessel CD68 expression was decreased, as were mRNA encoding several markers of M1-like macrophages: IL-1β: –44%; IL-6: –40%; MCP1: –25%; (P=0.02 for all) and M2-like macrophages: TGF-β1: –13% (P<0.001); ARG-1: –27% (P=0.03); IL-10: –23% (P=0.09). mRNA encoding IL-6 (100%; P<0.001) and TNF-α (P<0.05) was also reduced in endothelium of HDAdABCA1 arteries. Intimal/medial area ratio was slightly less in HDAdABCA1 carotids (–11%; P=0.06). Intimal lipid as well as macrophage and smooth muscle cell content were unchanged (P≥0.5 for all). Mass spectrometry showed no difference in unesterified or esterified cholesterol (P≥0.6 for both). Conclusion: ABCA1 overexpression in carotid endothelium reduced CD68 expression along with levels of several M1- and M2-like macrophage markers and inflammatory cytokines. ABCA1 overexpression had a small effect on intimal mass and did not reduce intimal lipid accumulation. ABCA1 overexpression in endothelium has mild atheroprotective effects.

Swapping the serotype: A novel helper-dependent adenoviral vector platform for in vivo HSC gene therapy

Swapping the serotype: A novel helper-dependent adenoviral vector platform for in vivo HSC gene therapy

Development and evaluation of helper dependent adenoviral vectors for inner ear gene delivery

Viral vector gene therapy is an attractive strategy to treat hearing loss. Since hearing loss is due to a variety of pathogenic signaling cascades in distinct cells, viral vectors that can express large or multiple genes in a cell-type specific manner are needed. Helper-dependent adenoviral vectors (HdAd) are safe viral vectors with a large packaging capacity (-36 kb). Despite the potential of HdAd, its use in the inner ear is largely unexplored. Therefore, to evaluate the utility of HdAd for inner ear gene therapy, we created two HdAd vectors that use distinct cellular receptors for transduction: HdAd Serotype Type 5 (HdAd5), the Coxsackie-Adenovirus Receptor (CAR) and a chimeric HdAd 5/35, the human CD46+ receptor (hCD46). We delivered these vectors through the round window (RW) or scala media in CBA/J, C57Bl6/J and hCD46 transgenic mice. Immunostaining in conjunction with confocal microscopy of cochlear sections revealed that multiple cell types were transduced using HdAd5 and HdAd 5/35 in all mouse models. Delivery of HdAd5 via RW in the C57Bl/6 J or CBA/J cochlea resulted in transduced mesenchymal cells of the peri‑lymphatic lining and modiolar region while scala media delivery resulted in transduction of supporting cells and inner hair cells. Hd5/35 transduction was CD46 dependent and RW delivery of HdAd5/35 in the hCD46 mouse model resulted in a similar transduction pattern as HdAd5 in the peri‑lymphatic lining and modiolar region in the cochlea. Our data indicate that HdAd vectors are promising vectors for use in inner ear gene therapy to treat some causes of hearing loss.

Stereotactic Delivery of Helper-dependent Adenoviral Viral Vectors at Distinct Developmental Time Points to Perform Age-dependent Molecular Manipulations of the Mouse Calyx of Held.

Synapses are specialized structures that enable neuronal communication, which is essential for brain function and development. Alterations in synaptic proteins have been linked to various neurological and neuropsychiatric disorders. Therefore, manipulating synaptic proteins in vivo can provide insight into the molecular mechanisms underlying these disorders and aid in developing new therapeutic strategies. Previous methods such as constitutive knock-out animals are limited by developmental compensation and off-target effects. The current approach outlines procedures for age-dependent molecular manipulations in mice using helper-dependent adenovirus viral vectors (HdAd) at distinct developmental time points. Using stereotactic injection of HdAds in both newborn and juvenile mice, we demonstrate the versatility of this method to express Cre recombinase in globular bushy cells of juvenile Rac1fl/fl mice to ablate presynaptic Rac1 and study its role in synaptic transmission. Separately, we overexpress CaV2 α1 subunits at two distinct developmental time points to elucidate the mechanisms that determine presynaptic CaV2 channel abundance and preference. This method presents a reliable, cost-effective, and minimally invasive approach for controlling gene expression in specific regions of the mouse brain and will be a powerful tool to decipher brain function in health and disease. Key features Virus-mediated genetic perturbation in neonatal and young adult mice. Stereotaxic injection allows targeting of brain structures at different developmental stages to study the impact of genetic perturbation throughout the development.

Advances in Gene Therapy Techniques to Treat LRRK2 Gene Mutation.

Leucine-rich repeat kinase 2 (LRRK2) gene mutation is an autosomal dominant mutation associated with Parkinson's disease (PD). Among LRRK2 gene mutations, the LRRK2 G2019S mutation is frequently involved in PD onset. Currently, diverse gene correction tools such as zinc finger nucleases (ZFNs), helper-dependent adenoviral vector (HDAdV), the bacterial artificial chromosome-based homologous recombination (BAC-based HR) system, and CRISPR/Cas9-homology-directed repair (HDR) or adenine base editor (ABE) are used in genome editing. Gene correction of the LRRK2 G2019S mutation has been applied whenever new gene therapy tools emerge, being mainly applied to induced pluripotent stem cells (LRRK2 G2019S-mutant iPSCs). Here, we comprehensively introduce the principles and methods of each programmable nuclease such as ZFN, CRISPR/Cas9-HDR or ABE applied to LRRK2 G2019S, as well as those of HDAdV or BAC-based HR systems used as nonprogrammable nuclease systems.

In vivo HSC transduction in rhesus macaques with an HDAd5/3+ vector targeting desmoglein 2 and transiently overexpressing cxcr4

AbstractWe developed a new in vivo hematopoietic stem cell (HSC) gene therapy approach that involves only IV injections and does not require myeloablation/conditioning and HSC transplantation. In this approach, HSCs are mobilized from the bone marrow into the peripheral bloodstream and transduced with IV injected helper-dependent adenovirus (HDAd) vectors. A fraction of transduced HSCs returns to the bone marrow and persists there long term. Here, we report desmoglein 2 (DSG2) as a new receptor that can be used for in vivo HSC transduction. HDAd5/3+ vectors were developed that use DSG2 as a high-affinity attachment receptor, and in vivo HSC transduction and safety after IV injection of an HDAd5/3+ vector expressing green fluorescent protein (GFP) in granulocyte colony-stimulating factor/AMD3100 (plerixafor)-mobilized rhesus macaques were studied. Unlike previously used CD46-targeting HDAd5/35++ vectors, HDAd5/3+ virions were not sequestered by rhesus erythrocytes and therefore mediated ∼10-fold higher GFP marking rates in primitive HSCs (CD34+/CD45RA–/CD90+ cells) in the bone marrow at day 7 after vector injection. To further increase the return of in vivo transduced, mobilized HSCs to the bone marrow, we transiently expressed cxcr4 in mobilized HSCs from the HDAd5/3+ vector. In vivo transduction with an HDAd5/3+GFP/cxcr4 vector at a low dose of 0.4 × 1012 viral particles/kg resulted in up to 7% of GFP-positive CD34+/CD45RA–/CD90+ cells in the bone marrow. This transduction rate is a solid basis for in vivo base or prime editing in combination with natural or drug-induced expansion of edited HSCs. Furthermore, our study provides new insights into HSC biology and trafficking after mobilization in nonhuman primates.

In Vivo Hematopoietic Stem Cell Gene Therapy for SARS-CoV2 Infection Using a Decoy Receptor.

While SARS-CoV2 vaccines have shown an unprecedented success, the ongoing emergence of new variants and necessity to adjust vaccines justify the development of alternative prophylaxis and therapy approaches. Hematopoietic stem cell (HSC) gene therapy using a secreted CoV2 decoy receptor protein (sACE2-Ig) would involve a one-time intervention resulting in long-term protection against airway infection, viremia, and extrapulmonary symptoms. We recently developed a technically simple and portable in vivo hematopoietic HSC transduction approach that involves HSC mobilization from the bone marrow into the peripheral blood stream and the intravenous injection of an integrating, helper-dependent adenovirus (HDAd5/35++) vector system. Considering the abundance of erythrocytes, in this study, we directed sACE2-Ig expression to erythroid cells using strong β-globin transcriptional regulatory elements. We performed in vivo HSC transduction of CD46-transgenic mice with an HDAd-sACE2-Ig vector. Serum sACE2-Ig levels reached 500–1,300 ng/mL after in vivo selection. At 22 weeks, we used genetically modified HSCs from these mice to substitute the hematopoietic system in human ACE2-transgenic mice, thus creating a model that is susceptible to SARS-CoV2 infection. Upon challenge with a lethal dose of CoV2 (WA-1), sACE2-Ig expressed from erythroid cells of test mice diminishes infection sequelae. Treated mice lost significantly less weight, had less viremia, and displayed reduced cytokine production and lung pathology. The second objective of this study was to assess the safety of in vivo HSC transduction and long-term sACE2-Ig expression in a rhesus macaque. With appropriate cytokine prophylaxis, intravenous injection of HDAd-sACE2-Ig into the mobilized animal was well tolerated. In vivo transduced HSCs preferentially localized to and survived in the spleen. sACE2-Ig expressed from erythroid cells did not affect erythropoiesis and the function of erythrocytes. While these pilot studies are promising, the antiviral efficacy of the approach has to be improved, for example, by using of decoy receptors with enhanced neutralizing capacity and/or expression of multiple antiviral effector proteins.

Safe and efficient in vivo hematopoietic stem cell transduction in nonhuman primates using HDAd5/35++ vectors

We tested a new in vivo hematopoietic stem cell (HSC) transduction/selection approach in rhesus macaques using HSC-tropic, integrating, helper-dependent adenovirus vectors (HDAd5/35++) designed for the expression of human γ-globin in red blood cells (RBCs) to treat hemoglobinopathies. We show that HDAd5/35++ vectors preferentially transduce HSCs in vivo after intravenous injection into granulocyte colony-stimulating factor (G-CSF)/AMD3100-mobilized animals and that transduced cells return to the bone marrow and spleen. The approach was well tolerated, and the activation of proinflammatory cytokines that are usually associated with intravenous adenovirus vector injection was successfully blunted by pre-treatment with dexamethasone in combination with interleukin (IL)-1 and IL-6 receptor blockers. Using our MGMTP140K-based in vivo selection approach, γ-globin+ RBCs increased in all animals with levels up to 90%. After selection, the percentage of γ-globin+ RBCs declined, most likely due to an immune response against human transgene products. Our biodistribution data indicate that γ-globin+ RBCs in the periphery were mostly derived from mobilized HSCs that homed to the spleen. Integration site analysis revealed a polyclonal pattern and no genotoxicity related to transgene integrations. This is the first proof-of-concept study in nonhuman primates to show that in vivo HSC gene therapy could be feasible in humans without the need for high-dose chemotherapy conditioning and HSC transplantation.

VikAD, a Vika site-specific recombinase-based system for efficient and scalable helper-dependent adenovirus production

Recombinant viral vectors have become integral tools for basic in vivo research applications. Helper-dependent adenoviral (HdAd) vectors have a large packaging capacity of ∼36 kb of DNA that mediate long-term transgene expression in vitro and in vivo. The large carrying capacity of HdAd enables basic research or clinical applications requiring the delivery of large genes or multiple transgenes, which cannot be packaged into other widely used viral vectors. Currently, common HdAd production systems use an Ad helper virus (HV) with a packaging signal (Ψ) that is flanked by either loxP or FRT sites, which is excised in producer cells expressing Cre or Flp recombinases to prevent HV packaging. However, these production systems prevent the use of HdAd vectors for genetic strategies that rely on Cre or Flp recombination for cell-type-specific expression. To overcome these limitations, we developed the VikAD production system, which is based on producer cells expressing the Vika recombinase and an HV that contains a Ψ flanked by vox sites. The availability of this production system will greatly expand the utility and flexibility of HdAd vectors for use in research applications to monitor and manipulate cellular activity with increased specificity.

Intranasal HD-Ad vaccine protects the upper and lower respiratory tracts of hACE2 mice against SARS-CoV-2

BackgroundThe ongoing COVID-19 pandemic has resulted in 185 million recorded cases and over 4 million deaths worldwide. Several COVID-19 vaccines have been approved for emergency use in humans and are being used in many countries. However, all the approved vaccines are administered by intramuscular injection and this may not prevent upper airway infection or viral transmission.ResultsHere, we describe a novel, intranasally delivered COVID-19 vaccine based on a helper-dependent adenoviral (HD-Ad) vector. The vaccine (HD-Ad_RBD) produces a soluble secreted form of the receptor binding domain (RBD) of the SARS-CoV-2 spike protein and we show it induced robust mucosal and systemic immunity. Moreover, intranasal immunization of K18-hACE2 mice with HD-Ad_RBD using a prime-boost regimen, resulted in complete protection of the upper respiratory tract against SARS-CoV-2 infection.ConclusionOur approaches provide a powerful platform for constructing highly effective vaccines targeting SARS-CoV-2 and its emerging variants.

Persistent Control of SIV Infection in Rhesus Macaques By Expressing a Highly Potent SIV Decoy Receptor after In Vivo HSC Transduction

Despite the success achieved by antiretroviral HIV-1 therapies, long-term and repeated drug administration is associated with toxicity, virus evasion and high cost. We aim to develop a gene therapy approach for persistent control against HIV-1 infection by delivering the transgene expressing a decoy receptor (eCD4-Ig) to hematopoietic stem cells (HSCs) by in vivo transduction. In this approach, HSCs are mobilized from the bone marrow into the peripheral blood stream and transduced with intravenously injected virus vectors. We use an integrating, helper-dependent adenovirus (HDAd5/35++) vector system that targets human CD46, a receptor that is abundantly expressed on primitive HSCs. Transgene integration is achieved by a hyperactive Sleeping Beauty transposase and transgene marking in peripheral blood cells can be increased by in vivo selection. The efficacy and safety of our in vivo HSC transduction/selection strategy has been previously demonstrated for the treatment of b-hemoglobinopathies, hemophilia A, and cancer in murine disease models. In non-human primates, we showed efficient transgene (g-globin) expression in peripheral red blood cells using this strategy. eCD4-Ig functions like a neutralizing antibody and shows broad neutralization spectrum against HIV-1, HIV-2 and SIV isolates. We have designed and produced an HDAd5/35++ vector expressing rhesus eCD4-Ig (rh-eCD4-Ig) from a constitutive and highly active EF1a promoter. In CD46-transgenic mice, over 50µg/mL eCD4-Ig in serum was measured after in vivo transduction and selection, with no obvious adverse events observed. Neutralization assay with serum samples showed that the produced eCD4-Ig effectively inhibited HIV-1 and SIV infection. We then performed studies in a rhesus macaque. After in vivo HSC transduction/selection of a rhesus macaque, eCD4-Ig serum levels were stable at 20-30 mg/ml. In vitro SIVmac239 neutralization assays using week 13 serum and recombinant eCD4-Ig protein determined that the IC50 of eCD4-Ig in rhesus serum is 1.0 mg/ml. This implies that the serum eCD4-Ig concentration at the time of SIVmac239 challenge was ~25-fold higher than the IC50. High-level eCD4-Ig expression had no clinical or hematological side effects. The first SIVmac239 challenge (20pg) was given on June 22 nd. Increasing rechallenge doses are currently injected monthly. So far, the viral load measured by quantitative RT-PCR is below detection limit. The animal is without symptoms and has normal lymphocyte/subset counts. Our study demonstrates an in vivo HSC transduction approach for potential long-term control of HIV-1 infection. DisclosuresKiem: VOR Biopharma: Consultancy; Ensoma Inc.: Consultancy, Current holder of individual stocks in a privately-held company; Homology Medicines: Consultancy. Lieber: Ensoma: Research Funding.

Next steps in the evolution of AAV vector characterization technologies

<div class="authors authors-lg"><div class="author"><div class="author-img"><img src="https://bioinsights.blob.core.windows.net/uploads/Eduard_picture copy.jpg" data-image="1"></div><div class="author-det"> <b>Eduard Ayuso</b> is Chief Technology Officer at DiNAQOR (Zürich, Switzerland), a genetic medicine platform company focused on addressing severe inherited cardiac diseases. Dr Ayuso is an expert in the field of gene therapy using viral vector platforms, including adenoviral vectors, helper-dependent adenoviral vectors, and adeno-associated vectors (AAV), as well as their design, manufacture, and purification. With over two decades of academic and industry consulting experience, he has made significant contributions to the field of in vivo gene transfer in small and large animal models of diseases, as well as AAV vector development and analytics. Dr Ayuso previously served as Head of Innovative Vectorology at the French National Institute of Health and Medical Research (INSERM), and as the Scientific Director of the Translational Vector Core unit at University of Nantes. Since 2018, he has served as vice president of the French Society for Gene and Cell Therapy. He is also a member of the Translational Science and Drug Product Development committee of the American Society for Gene and Cell Therapy. Dr Ayuso earned his PhD in Biochemistry and Molecular Biology and his degree in Veterinary Medicine from Autonomous University of Barcelona. </div></div></div>

In Vivo HSC Transduction and Selection Results in Long-Term, High-Level Expression of Human Gamma Globin in Peripheral Blood Erythrocytes of Mice

Current protocols for HSC gene therapy, involving the transplantation of ex vivo lentivirus vector-transduced HSCs into myeloablated recipients, are complex and not without risk for the patient. We have developed a new approach for in vivo gene delivery into HSCs that does not require myeloablation and HSC transplantation. It involves injections of G-CSF/AMD3100 to mobilize HSCs from the bone marrow into the peripheral blood stream and the intravenous injection of an integrating, helper-dependent adenovirus (HDAd5/35++) vector system. Transgene integration is achieved (in a random pattern) using a hyperactive Sleeping Beauty transposase (SB100x). We demonstrated in adequate mouse models, using GFP as a transgene, that primitive HSCs transduced in the periphery home back to the bone marrow where they persist and stably express GFP long-term (Blood 128:2206 (2016). To achieve high level transgene marking in differentiated peripheral blood cells, we combined our in vivo HSC transduction approach with in vivo selection of transduced HSCs using the mgtmP140K mutant and low dose O6BG/BCNU treatment. Here were tested the potential of this approach for the therapy of hemoglobinopathies. Our HDAd5/35++ vector contained the human-gamma globin gene under the control of a 6kb version of the beta-globin LCR (containing HS1 to HS4 and the beta globin promoter) and the mgmtP140K gene under the control of the PGK promoter. Mobilized immunocompetent hCD46 transgenic mice were injected IV with the integrating vector system and subjected to three rounds of treatment with low-dose O6BG/BCNU followed by immunosuppression to avoid immune responses against the human transgene product. The procedure was safe and resulted in stable human gamma globin expression in >95% of erythrocytes in the peripheral blood of in vivo transduced cells mice. Human gamma globin levels were 100% of endogenous mouse beta globin levels in erythrocytes measured by HPLC. Analysis of bone marrow at week 16 after in vivo transduction showed that >60% of bone marrow LSK cells contained the integrated transgene cassette and that the beta globin LCR efficiently restricted gamma globin expression to erythroid cells. After transplantation of unsorted, lineage-depleted bone marrow cells into lethally irradiated recipients, long-term gamma-globin expression at levels seen in the primary in vivo treated mice was demonstrated. The efficacy of our approach achieved in this study indicates that it would be curative in thalassemia models or patients. Our new approach avoids the collection of HSCs, their transduction ex vivo, and subsequent transplantation into myeloablated recipients. It therefore could greatly simplify HSC gene therapy of common diseases. DisclosuresKiem:Rocket Pharmaceuticals: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

A “Shot in the Arm” for Sickle Cell Disease

A “Shot in the Arm” for Sickle Cell Disease

A helper-dependent adenoviral vector rescues CFTR to wild-type functional levels in cystic fibrosis epithelial cells harbouring class I mutations.

Cystic fibrosis (CF) is a genetic disorder affecting multiple organs, including the pancreas, hepatobiliary system and reproductive organs; however, lung disease is responsible for the majority of morbidity and mortality. Management of CF involves CF transmembrane conductance regulator (CFTR) modulator agents including corrector drugs to augment cellular trafficking of mutant CFTR as well as potentiators that open defective CFTR channels. These therapies are poised to help most individuals with CF, with the notable exception of individuals with class I mutations where full-length CFTR protein is not produced. For these mutations, gene replacement has been suggested as a potential solution.In this work, we used a helper-dependent adenoviral vector (HD-CFTR) to express CFTR in nasal epithelial cell cultures derived from CF subjects with class I CFTR mutations.CFTR function was significantly restored in CF cells by HD-CFTR and reached healthy control functional levels as detected by Ussing chamber and membrane potential (FLIPR) assay. A dose-response relationship was observed between the amount of vector used and subsequent functional outcomes; small amounts of HD-CFTR were sufficient to correct CFTR function. At higher doses, HD-CFTR did not increase CFTR function in healthy control cells above baseline values. This latter observation allowed us to use this vector to benchmark in vitro efficacy testing of CFTR-modulator drugs.In summary, we demonstrate the potential for HD-CFTR to inform in vitro testing and to restore CFTR function to healthy control levels in airway cells with class I or CFTR nonsense mutations.

Overcoming Immunological Challenges to Helper-Dependent Adenoviral Vector-Mediated Long-Term CFTR Expression in Mouse Airways.

Cystic Fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and CF patients require life-long treatment. Although CFTR modulators show a great potential for treating most CF patients, some individuals may not tolerate the treatment. In addition, there is no effective therapy for patients with some rare CFTR mutations, such as class I CF mutations, which lead to a lack of CFTR protein production. Therefore, other therapeutic strategies, such as gene therapy, have to be investigated. Currently, immune responses to gene therapy vectors and transgene products are a major obstacle to applying CF gene therapy to clinical applications. In this study, we examined the effects of cyclophosphamide on the modulation of host immune responses and for the improvement of the CFTR transgene expression in the repeated delivery of helper-dependent adenoviral (HD-Ad) vectors to mouse lungs. We have found that cyclophosphamide significantly decreased the expression of T cell genes, such as CD3 (cluster of differentiation 3) and CD4, and reduced their infiltration into mouse lung tissues. We have also found that the levels of the anti-adenoviral antibody and neutralizing activity as well as B-cell infiltration into the mouse lung tissues were significantly reduced with this treatment. Correspondingly, the expression of the human CFTR transgene has been significantly improved with cyclophosphamide administration compared to the group with no treatment. These data suggest that the sustained expression of the human CFTR transgene in mouse lungs through repeated vector delivery can be achieved by transient immunosuppression.

Transient immunosuppression blocks iBALT formation in the lung induced by helper-dependent adenoviral vector during gene delivery

Abstract Lung is an immune sensitive organ with presence of all immune cells at the frontline of immunity. Under chronic infection conditions, inducible Bronchus-associated lymphoid tissue (iBALT) can be formed to defend foreign infectious organisms. We wonder whether viral vectors for gene therapy could induce iBALT formation and whether this process can be controlled through immune modulation. To test these ideas, we repeatedly administered a helper-dependent adenoviral (HD-Ad) vector to mouse lungs and examined iBALT formation. HD-Ad vectors are based on adenovirus but with all viral coding sequences deleted and is one option for large gene delivery. In our previous studies, HD-Ad vectors have exhibited high in vivo efficiency in transduction of mouse, rabbit and pig airway epithelial cells, including stem/progenitor cells such as basal cells. HD-Ad vectors expressing therapeutic gene encoding cystic fibrosis transmenbrane conductance regulator (CFTR) rescue CFTR function in CF patient primary cells. In our mouse studies, we found that HD-Ad vectors induced iBALT formation and antibody production. We used cyclophosphamide or HD-Ad expressing IL-10 to modulate the host immune responses and to examine the effects on iBALT formation. Comparing with control group (HD-Ad vectors only), the mice treated with cyclophosphamide or IL-10 expressing HD-Ad vector showed a significant reduction of iBALT formation and infiltration of inflammatory cells, including CD4+ and B220+ as well as production of anti-Ad antibodies and neutralizing antibodies. Our results indicate that transient modulation significantly mitigated immunopathology and extended transgene expression in mouse airways following readministration of HD-Ad vectors.

Danio Rerio as Model Organism for Adenoviral Vector Evaluation.

Viral vector use is wide-spread in the field of gene therapy, with new clinical trials starting every year for different human pathologies and a growing number of agents being approved by regulatory agencies. However, preclinical testing is long and expensive, especially during the early stages of development. Nowadays, the model organism par excellence is the mouse (Mus musculus), and there are few investigations in which alternative models are used. Here, we assess the possibility of using zebrafish (Danio rerio) as an in vivo model for adenoviral vectors. We describe how E1/E3-deleted adenoviral vectors achieve efficient transduction when they are administered to zebrafish embryos via intracranial injection. In addition, helper-dependent (high-capacity) adenoviral vectors allow sustained transgene expression in this organism. Taking into account the wide repertoire of genetically modified zebrafish lines, the ethical aspects, and the affordability of this model, we conclude that zebrafish could be an efficient alternative for the early-stage preclinical evaluation of adenoviral vectors.

Adenovirus vectors in hematopoietic stem cell genome editing.

Genome editing of hematopoietic stem cells (HSCs) represents a therapeutic option for a number of hematological genetic diseases, as HSCs have the potential for self-renewal and differentiation into all blood cell lineages. This review presents advances of genome editing in HSCs utilizing adenovirus vectors as delivery vehicles. We focus on capsid-modified, helper-dependent adenovirus vectors that are devoid of all viral genes and therefore exhibit an improved safety profile. We discuss HSC genome engineering for several inherited disorders and infectious diseases including hemoglobinopathies, Fanconi anemia, hemophilia, and HIV-1 infection by exvivo and invivo editing in transgenic mice, nonhuman primates, as well as in human CD34+ cells. Mechanisms of therapeutic gene transfer including episomal expression of designer nucleases and base editors, transposase-mediated random integration, and targeted homology-directed repair triggered integration into selected genomic safe harbor loci are also reviewed.