Helper-dependent Adenovirus Research Articles

CCN4 (WISP-1) reduces apoptosis and atherosclerotic plaque burden in an ApoE mouse model

Background and aimsCCN4/WISP-1 regulates various cell behaviours that contribute to atherosclerosis progression, including cell adhesion, migration, proliferation and survival. We therefore hypothesised that CCN4 regulates the development and progression of atherosclerotic plaques. MethodsWe used a high fat fed ApoE−/− mouse model to study atherosclerotic plaque progression in the brachiocephalic artery and aortic root. In protocol 1, male ApoE−/− mice with established plaques were given a CCN4 helper-dependent adenovirus to see the effect of treatment with CCN4, while in protocol 2 male CCN4−/−ApoE−/− were compared to CCN4+/+ApoE−/− mice to assess the effect of CCN4 deletion on plaque progression. ResultsCCN4 overexpression resulted in reduced occlusion of the brachiocephalic artery with less apoptosis, fewer macrophages, and attenuated lipid core size. The amount of plaque found on the aortic root was also reduced. CCN4 deficiency resulted in increased apoptosis and occlusion of the brachiocephalic artery as well as increased plaque in the aortic root. Additionally, in vitro cells from CCN4−/−ApoE−/− mice had higher apoptotic levels. CCN4 deficiency did not significantly affect blood cholesterol levels or circulating myeloid cell populations. ConclusionsWe conclude that in an atherosclerosis model the most important action of CCN4 is the effect on cell apoptosis. CCN4 provides pro-survival signals and leads to reduced cell death, lower macrophage number, smaller lipid core size and reduced atherosclerotic plaque burden. As such, the pro-survival effect of CCN4 is worthy of further investigation, in a bid to find a therapeutic for atherosclerosis.

Mesenchymal stromal cells protect combined oncolytic and helper-dependent adenoviruses from humoral immunity

Systemic delivery of oncolytic and immunomodulatory adenoviruses may be required for optimal effects on human malignancies. Mesenchymal stromal cells (MSCs) can serve as delivery systems for cancer therapeutics due to their ability to transport and shield these agents whilst homing to tumors. We now use MSCs to deliver a clinically validated binary oncolytic and helper dependent adenovirus combination (CAdVEC) to tumor cells. We show successful oncolysis and helper-dependent virus function in tumor cells even in the presence of plasma from adenovirus seropositive donors. In both two- and three-dimensional cultures, CAdVEC function is eliminated even at high dilutions of seropositive plasma but is well sustained when CAdVEC is delivered by MSCs. These results provide a robust in vitro model to measure oncolytic and helper dependent virus spread and demonstrate a beneficial role of using MSCs for systemic delivery of CAdVEC even in the presence of a neutralizing humoral response.

Development of adenoviral vectors that transduce Purkinje cells and other cerebellar cell-types in the cerebellum of a humanized mouse model

Viral vector gene therapy has immense promise for treating central nervous system (CNS) disorders. Although adeno-associated virus vectors (AAVs) have had success, their small packaging capacity limits their utility to treat the root cause of many CNS disorders. Adenoviral vectors (Ad) have tremendous potential for CNS gene therapy approaches. Currently, the most common vectors utilize the Group C Ad5 serotype capsid proteins, which rely on the Coxsackievirus-Adenovirus receptor (CAR) to infect cells. However, these Ad5 vectors are unable to transduce many neuronal cell types that are dysfunctional in many CNS disorders. The human CD46 (hCD46) receptor is widely expressed throughout the human CNS and is the primary attachment receptor for many Ad serotypes. Therefore, to overcome the current limitations of Ad vectors to treat CNS disorders, we created chimeric first generation Ad vectors that utilize the hCD46 receptor. Using a "humanized" hCD46 mouse model, we demonstrate these Ad vectors transduce cerebellar cell types, including Purkinje cells, that are refractory to Ad5 transduction. Since Ad vector transduction properties are dependent on their capsid proteins, these chimeric first generation Ad vectors open new avenues for high-capacity helper-dependent adenovirus (HdAd) gene therapy approaches for cerebellar disorders and multiple neurological disorders.

Prime editing-mediated correction of the CFTR W1282X mutation in iPSCs and derived airway epithelial cells.

A major unmet need in the cystic fibrosis (CF) therapeutic landscape is the lack of effective treatments for nonsense CFTR mutations, which affect approximately 10% of CF patients. Correction of nonsense CFTR mutations via genomic editing represents a promising therapeutic approach. In this study, we tested whether prime editing, a novel CRISPR-based genomic editing method, can be a potential therapeutic modality to correct nonsense CFTR mutations. We generated iPSCs from a CF patient homozygous for the CFTR W1282X mutation. We demonstrated that prime editing corrected one mutant allele in iPSCs, which effectively restored CFTR function in iPSC-derived airway epithelial cells and organoids. We further demonstrated that prime editing may directly repair mutations in iPSC-derived airway epithelial cells when the prime editing machinery is efficiently delivered by helper-dependent adenovirus (HDAd). Together, our data demonstrated that prime editing may potentially be applied to correct CFTR mutations such as W1282X.

Apolipoprotein A-I vascular gene therapy reduces vein-graft atherosclerosis

Coronary artery venous bypass grafts typically fail because of atherosclerosis driven by lipid and macrophage accumulation. Therapy for vein-graft atherosclerosis is limited to statin drugs, which are only modestly effective. We hypothesized that transduction of vein-graft endothelium of fat-fed rabbits with a helper-dependent adenovirus expressing apolipoprotein AI (HDAdApoAI) would reduce lipid and macrophage accumulation. Fat-fed rabbits received bilateral external jugular vein-to-carotid artery interposition grafts. Four weeks later, one graft per rabbit (n= 23 rabbits) was infused with HDAdApoAI and the contralateral graft with HDAdNull. Grafts were harvested 12weeks later. Paired analyses of grafts were performed, with vein graft cholesterol, intimal lipid, and macrophage content as the primary endpoints. HDAd genomes were detected in all grafts. APOAI mRNA was median 63-fold higher in HDAdApoAI grafts versus HDAdNull grafts (p<0.001). HDAdApoAI grafts had a mean 15% lower total cholesterol (by mass spectrometry; p= 0.003); mean 19% lower intimal lipid (by oil red O staining; p= 0.02); and mean 13% lower expression of the macrophage marker CD68 (by reverse transcriptase-mediated quantitative PCR; p= 0.008). Invivo transduction of vein-graft endothelium achieves persistent APOAI expression and reduces vein-graft cholesterol, intimal lipid, and CD68 expression. Vascular gene therapy with APOAI haspromise for preventing vein-graft failure caused by atherosclerosis.

Beyond Busulfan: Building a Better Conditioner for Bone Marrow Transplantation

Beyond Busulfan: Building a Better Conditioner for Bone Marrow Transplantation

HDAd6/35++ - A new helper-dependent adenovirus vector platform for in vivo transduction of hematopoietic stem cells

In previous studies, we achieved safe and efficient invivo hematopoietic stem cell (HSC) transduction in mobilized mice and macaques with intravenously injected helper-dependent adenovirus HDAd5/35++ vectors. These vectors are derivatives of serotype Ad5-containing CD46-affinity enhanced Ad35 fiber knob domains. Considering the impact of anti-Ad5/HDAd5/35++ neutralizing serum antibodies present in the human population, we generated HSC-retargeted HDAd6/35++ vectors derived from serotype 6. We found a lower prevalence and titers of serum anti-HDAd6/35++ in human samples compared with HDAd5/35++. HDAd6/35++ vectors efficiently transduced human and rhesus CD34+ cells invitro. Intravenous injection of HDAd5/35++-GFP or HDAd6/35++-GFP vectors after G-CSF/AMD3100 mobilization of mice with established human hematopoiesis or human CD46 transgenic mice resulted in comparable GFP marking rates in HSCs in the bone marrow and spleen. In long-term invivo HSC transduction and selection studies with integrating vectors, stable GFP expression in >75% of PBMCs was show for both vectors. In contrast with HDAd5/35++, undesired transduction of hepatocytes was minimal with HDAd6/35++. Furthermore, HDAd6/35++ allowed for efficient invivo HSC transduction in Ad5-pre-immune mice. These features, together with the straightforward production of HDAd6/35++ vectors at high yield, make this new HDAd vector platform attractive for clinical translation of the invivo approach.

Abstract P3-06-04: Treatment of Metastatic Breast Cancer with Multipotent Oncolytic/Helper Adenovirus CAdVEC

Abstract Background: Metastatic breast cancer (MBC) which causes significant morbidity and mortality worldwide is in need of more effective treatment regimens. In combination with chemotherapy, anti-PD1 antibody pembrolizumab has been shown to prolong progression-free survival (PFS) of patients with triple-negative subtype MBC (TN-MBC), however, its efficacy remains low for the other 80% of patients with MBC. MBC’s heterogenous pattern of immune infiltration and expression make it challenging to treat with single immunotherapeutic agents such as pembrolizumab and successful immunotherapy must therefore target multiple pathways. To augment antitumor host immune responses during treatment, studies have examined adjunct agents such as “oncolytic” adenovirus (OAds), which are vectors that preferentially replicate in and lyse tumor cells leading to the activation of host immunity. OAds have been tested in a myriad of clinical trials with the hope to enhance host immune activation but those trials have shown limited successes. Methods: To overcome the multiple immunogenic barriers in solid tumors, our group developed a binary oncolytic/helper-dependent adeno-immunotherapy (CAdVEC). The first generation CAdVEC (CAdTrio) contains an OAd and a “helper-dependent” adenovirus (HDAd) that produces immunostimulant molecules including interleukin (IL)-12p70 and anti-PD-L1 antibody. Based on successful results using animal models, a first-in-human Phase 1 study with CAdTrio was conducted among patients with all solid tumors (NCT03740256). Four patients with MBC were enrolled in the virus dose-escalation phase of the trial and received an intra-tumor injection of CAdTrio. Given the novelty of this binary agent, starting dose of CAdTrio was more than 2-logs lower than that used in other OAd trials. Three patients received dose level (DL) 1 and one patient received DL2. All patients also received pembrolizumab 6 weeks after the virus injection. The primary endpoint for this phase I dose escalation was dose-limiting toxicities (DLT). Secondary outcomes included overall response rates (ORR), disease control rate (DCR), PFS, overall survival (OS), and treatment-related adverse events. Results: No patients developed DLT. The most common toxicities were fever, fatigue and pain around the injection site, but none were greater than grade 2. No significant elevation in liver enzymes were observed. Three of the four patients had partial response (PR). One patient progressed after ten weeks of stable disease and passed away. The three patients with PR received pembrolizumab within 7 weeks of CAdVEC injection. Analysis of injected tissues prior to pembrolizumab treatment showed that CAdTrio repolarized the tumor microenvironment toward immune activity by increasing the number of infiltrating Th1 immune cells, leading to responses in some treated tumors and even in one distant metastasis, demonstrating the potent systemic immune response to local CAdTrio treatment in patients with MBC. Conclusions: Our study demonstrated that intra-tumor injection with CAdTrio was safe and effective in patients with MBC but the significance of the results was limited by the small sample size. An MBC dedicated phase II trial is planned to be conducted to fully evaluate the efficacy and safety of CAdVEC treatment and to further elucidate mechanisms of resistance/sensitivity among patients with MBC. Citation Format: Natalie Chen, Daniel Wang, Caroline E. Porter, Amanda Rosewell Shaw, Catherine R. Robertson, Mae L. Woods, Ya Xu, Greyson Biegert, Alphi Kuriakose, Tao Wang, Bambi J. Grilley, Helen Heslop, Malcolm K. Brenner, Masataka Suzuki, Bora Lim. Treatment of Metastatic Breast Cancer with Multipotent Oncolytic/Helper Adenovirus CAdVEC [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-06-04.

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.

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.

Abstract 430: GFP-tagging Of Rabbit ApoAI Decreases Expression And Cholesterol-efflux Activity

Introduction: Gene therapy using a helper-dependent adenovirus (HDAd) to express rabbit apolipoprotein (apo) AI in carotids of fat-fed rabbits reduces atherosclerosis. We hypothesize that transgenic apoAI promotes reverse cholesterol transport from the artery wall; however, this could not be tested because the transgenic rabbit apoAI is indistinguishable from endogenous rabbit apoAI. We therefore constructed an HDAd expressing GFP-tagged rabbit apoAI. Methods: We constructed HDAdGFPapoAI by fusing the GFP sequence to the N-terminus of the rabbit APOA1 gene with a short linker. Bovine aortic endothelial cells (BAEC) were transduced with HDAdGFPapoAI, washed, and incubated in serum-free medium for 24h. Conditioned medium (CM) was collected and fluorescence measured. Cell extracts and CM were analyzed by western blotting for protein size, expression level, and presence of GFP and apoAI antigens. CM cholesterol-efflux activity was measured using cholesterol-loaded BHK cells expressing ABCA1. Results: CM of HDAdGFPapoAI-transduced BAEC had green fluorescence and contained a protein of the size predicted for the GFP-apoAI fusion (~56 kDa), which was detected with antibodies to both GFP and apoAI. A smaller protein (~30 kDa) with GFP and apoAI antigens was also detected. Compared to a vector expressing WT rabbit apoAI, GFP-tagging decreased expression of apoAI protein by ~50% and the percentage of secreted apoAI from 55% to 13%. In combination, this reduced secreted GFP-apoAI to only 12% of levels of secreted WT apoAI. While CM containing WT rabbit apoAI strongly stimulated cholesterol efflux from BHK cells (46%), GFP-apoAI-containing CM did not promote cholesterol efflux compared to CM from untransduced BAEC (7.8% vs 7.4% efflux), even when present in CM at a concentration similar to that of WT apoAI. Conclusion: GFP-rabbit apoAI protein has reduced expression and secretion (likely due to misfolding/degradation) and does not promote cholesterol efflux (n-terminal GFP may disrupt the phospholipid/cholesterol organization needed for efflux via ABCA1 to this dynamic apoprotein). It will not be useful to investigate mechanisms of atheroprotection by vessel wall-expressed apoAI. Alternatives include use of a smaller tag or expression of human apoAI.

Abstract 444: Apolipoprotein A-I (ApoAI) Vascular Gene Therapy Protects Against Vein-Graft Atherosclerosis

Introduction: Coronary artery venous bypass grafts typically fail because of accelerated atherosclerosis, driven by lipid infiltration and by accumulation of macrophages and other cells. We previously showed that transduction of carotid artery endothelium of fat-fed rabbits with a helper-dependent adenovirus expressing apolipoprotein AI (HDAdApoAI) reduced atherosclerotic lesion size and decreased intimal lipid and macrophage accumulation. We hypothesized that HDAdApoAI transduction of rabbit vein graft endothelium would reduce lipid and macrophage accumulation. Methods: Fat-fed rabbits received bilateral external jugular vein to carotid artery interposition grafts. Four wks later, one graft per rabbit was infused with HDAdApoAI and the other with HDAdNull (a non-expressing HDAd). Grafts were harvested 12 wks after HDAd infusion. Paired analysis was performed in all rabbits with bilateral patent grafts (n=22). Graft hemodynamics were measured at vector-infusion and harvest surgeries. In vivo graft diameters were measured before vector infusion and harvest with ultrasound. Grafts were cut into 8 segments, for histology and biochemical analyses. Results: Mean plasma cholesterol levels were 400-600 mg/dL. Graft patency rates were 89% after vein grafting and 100% after the vector-infusion surgery. Lumen diameter and hemodynamics did not differ between HDAdApoAI and HDAdNull-transduced grafts. HDAd genomes were detected in all vein grafts. High levels of apoAI mRNA were detected in 100% of HDAdApoAI-treated vein grafts (mean 155-fold higher than in HDAdNull grafts). HDAdApoAI-treated grafts had 25% lower cholesterol (by mass spectrometry; P=0.03) and 17% less median CD68 mRNA (by RT-qPCR; P=0.008; CD68 is expressed by macrophages and lipid-loaded SMC). No significant differences were detected in mRNA encoding several inflammatory cytokines and inflammation-associated proteins. There were nominal decreases in total intimal area (16%, P=0.9), percent stenosis (4%, P=0.08), and in intimal area positive for Oil Red O (11%, P=0.3), macrophages (11%, P=0.7), and VCAM-1 (8%, P=0.8). Conclusion: HDAdApoAI transduction of vein graft endothelium reduces some markers of atherosclerosis, potentially by stimulating cholesterol efflux.

Efficacy and Safety of FX201, a Novel Intra-Articular IL-1Ra Gene Therapy for Osteoarthritis Treatment, in a Rat Model.

Osteoarthritis (OA) is a disabling, degenerative disease characterized by progressive cartilage and bone damage. There remains a need for local therapies that, following a single injection, can provide long-term pain relief and functional improvement and potentially delay disease progression. FX201 is a novel, intra-articular (IA), interleukin-1 receptor antagonist (IL-1Ra) gene therapy in development for the treatment of OA. In this study, we assessed the efficacy, biodistribution, and safety of helper-dependent adenovirus (HDAd)-ratIL-1Ra, the rat surrogate of FX201, and the biodistribution of FX201, in the anterior cruciate ligament transection (ACLT) rat OA model. A single IA injection of HDAd-ratIL-1Ra administered 7 days post-ACLT mitigated OA-related changes to cartilage, bone, and the synovial membrane at week 12 following surgery. Furthermore, FX201 and HDAd-ratIL-1Ra persisted for at least 92 days in the injected joint and proximal tissues with minimal evidence of vector spreading peripherally. Finally, HDAd-ratIL-1Ra showed a favorable safety profile without any local or systemic adverse effects. In conclusion, HDAd-ratIL-1Ra demonstrated local therapeutic and disease-modifying effects and was well tolerated, supporting further clinical development of FX201.

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.

Chimeric Helper-Dependent Adenoviruses Transduce Retinal Ganglion Cells and Müller Cells in Human Retinal Explants.

Purpose: Despite numerous recent advances in retinal gene therapy using adeno-associated viruses (AAVs) as delivery vectors, there remains a crucial need to identify viral vectors with the ability to transduce specific retinal cell types and that have a larger carrying capacity than AAV. In this study, we evaluate the retinal tropism of 2 chimeric helper-dependent adenoviruses (HDAds), helper-dependent adenovirus serotype 5 (HDAd5)/3 and HDAd5/35, both ex vivo using human retinal explants and in vivo using rats. Methods: We transduced cultured human retinal explants with HDAd5/3 and HDAd5/35 carrying an eGFP vector and evaluated tropism and transduction efficiency using immunohistochemistry. To assess in vivo transduction efficiency, subretinal injections were performed in wild-type Sprague-Dawley rats. For both explants and subretinal injections, we delivered 10 μL (1 × 106 vector genomes/mL) and assessed tropism at 7- and 14-days post-transduction, respectively. Results: HDAd5/3 and HDAd5/35 both transduced human retinal ganglion cells (RGCs) and Müller cells, but not photoreceptors, in human retinal explants. However, subretinal injections in albino rats resulted in transduction of the retinal pigmented epithelium only, highlighting species-specific differences in retinal tropism and the value of a human explant model when testing vectors for eventual human gene therapy. Conclusions: Chimeric HDAds are promising candidates for the delivery of large genes, multiple genes, or neuroprotective factors to Müller cells and RGCs. These vectors may have utility for targeted therapy of neurodegenerative diseases primarily involving retinal ganglion or Müller cell types, such as glaucoma or macular telangiectasia type 2.

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.