Helper-dependent Adenovirus Research Articles

Single-dose MGTA-145/plerixafor leads to efficient mobilization and in vivo transduction of HSCs with thalassemia correction in mice

We have developed an in vivo hemopoietic stem cell (HSC) gene therapy approach without the need for myelosuppressive conditioning and autologous HSC transplantation. It involves HSC mobilization and IV injection of a helper-dependent adenovirus HDAd5/35++ vector system. The current mobilization regimen consists of granulocyte colony-stimulating factor (G-CSF) injections over a 4-day period, followed by the administration of plerixafor/AMD3100. We tested a simpler, 2-hour, G-CSF–free mobilization regimen using truncated GRO-β (MGTA-145; a CXCR2 agonist) and plerixafor in the context of in vivo HSC transduction in mice. The MGTA-145+plerixafor combination resulted in robust mobilization of HSCs. Importantly, compared with G-CSF+plerixafor, MGTA-145+plerixafor led to significantly less leukocytosis and no elevation of serum interleukin-6 levels and was thus likely to be less toxic. With both mobilization regimens, after in vivo selection with O6-benzylguanine (O6BG)/BCNU, stable GFP marking was achieved in >90% of peripheral blood mononuclear cells. Genome-wide analysis showed random, multiclonal vector integration. In vivo HSC transduction after mobilization with MGTA-145+plerixafor in a mouse model for thalassemia resulted in >95% human γ-globin+ erythrocytes at a level of 36% of mouse β-globin. Phenotypic analyses showed a complete correction of thalassemia. The γ-globin marking percentage and level were maintained in secondary recipients, further demonstrating that MGTA145+plerixafor mobilizes long-term repopulating HSCs. Our study indicates that brief exposure to MGTA-145+plerixafor may be advantageous as a mobilization regimen for in vivo HSC gene therapy applications across diseases, including thalassemia and sickle cell disease.

A “Shot in the Arm” for Sickle Cell Disease

A “Shot in the Arm” for Sickle Cell Disease

In Vivo HSC Gene Therapy for Hemoglobinopathies: A Proof of Concept Evaluation in Rhesus Macaques

Current gene therapy or genome editing studies for hemoglobinopathies require highly sophisticated medical facilities to perform hematopoietic stem cell collections / selections and genetic modifications. In addition, patients receive high-dose chemotherapy to facilitate engraftment of gene-modified cells. Thus, current gene therapy protocols will not be accessible to most patients suffering from hemoglobinopathies. Here we describe a highly portable and scalable approach using in vivo hematopoietic stem cell (HSC) gene therapy to potentially overcome these limitations. The central idea of our in vivo HSC gene therapy approach is to mobilize HSCs from the bone marrow, and while they circulate at high numbers in the periphery, transduce them with an intravenously injected HSC-tropic, helper-dependent adenovirus HDAd5/35++ gene transfer vector system. Transduced cells return to the bone marrow where they persist long-term. Transgene integration is either achieved by a Sleeping Beauty transposase (SB100x) in a random pattern or by homology-directed-repair into a safe genomic harbor site. Currently, an in vivo selection system (involving the mgmtP140K gene/low-dose O6BG/BCNU) is employed to achieve 80-100% marking levels in peripheral blood cells. We demonstrated safety and efficacy of our approach in mouse models for thalassemia intermedia, Sickle Cell Disease, and hemophilia A, where we achieved a phenotypic correction. We now present data in 3 rhesus macaques. We show that treatment with G-CSF/AMD3100 resulted in efficient HSC mobilization into the blood circulation and subsequent intravenous injection of the HDAd5/35++ vector system (total 1-3 x1012 vp/kg, in two doses) was well tolerated. The longest follow up thus far is 24 weeks after in vivo HSC transduction with a human-gamma-globin expressing HDAd5/35++ vector. After in vivo selection with O6BG plus low dose (10 to 20 mg/m2) of BCNU, a dose that is up to 100-fold lower than what is used for autologous transplantation protocols, gamma-globin marking in peripheral red blood cells rose to ~90% and was stable for the duration of the study (see Figure). gamma-globin levels in red blood cells were ~18% of adult alpha1-globin (by HPLC). No abnormalities in genome and transcriptome analyses of animal #1 were found at the time of scheduled necropsy. We show that a new prophylaxis regimen (dexamethasone, IL-6R, IL-1bR antagonists, saline bolus IV) was able to mitigate all side effects associated with intravenous HDAd5/35++ vector administration. Analysis of day 3 bone marrow showed 30% transduced HSCs. Vector DNA biodistribution studies demonstrated very low or absent transduction of most tissues (including testes and CNS). Analysis of bone marrow showed efficient, preferential HSC transduction and re-homing of transduced CD34+/CD90+ cells to the bone marrow. At week 4, about 5% of progenitor colony-forming cells demonstrated stable transduction with integrated vector, and this frequency increased after starting the in vivo selection. The level of human mgmtP140K mRNA expression in PBMCs also increased after in vivo selection. In summary: Using a new and optimized prophylaxis regimen intravenous delivery of HDAd5/35++ was very well tolerated without any cytokine activation. We saw efficient transduction of HSCs and efficient in vivo selection of transduced progenitors with low dose O6BG/BCNU. This is the first proof-of-concept study that in vivo HSC gene therapy could be feasible in humans without the need of high-dose chemotherapy conditioning and without the need for highly specialized medical facilities. This approach would provide a major advance for the gene therapy and genome editing field and allow the necessary portability and accessibility to reach patients in places with limited medical resources. Figure 1 Disclosures Radtke: Forty Seven INC: Consultancy. Kiem:Umoja: Membership on an entity's Board of Directors or advisory committees; Rocket Pharma: Membership on an entity's Board of Directors or advisory committees; Vor Biopharma: Membership on an entity's Board of Directors or advisory committees; Enochian: Membership on an entity's Board of Directors or advisory committees; CSL: Consultancy; Magenta Therapeutics: Consultancy; Homology Medicines: Membership on an entity's Board of Directors or advisory committees. Lieber:Ensoma, Inc: Consultancy, Research Funding.

Mgta-145 / Plerixafor-Mediated HSC Mobilization and Intravenous HDAd5/35++ Vector Injection into Mice Allows for Efficient In Vivo HSC Transduction and Stable Gene Marking in Peripheral Blood Cells of CD46-Transgenic and Thalassemia Mice

Background. In vivo hematopoietic stem cell (HSC) gene therapy represents a simpler approach to treating hemoglobinopathies without the need for myelosuppressive conditioning and autologous HSC transplantation. We developed a helper dependent adenovirus (HDAd5/35++)-based platform that enables efficient in vivo transduction of mobilized HSCs via CD46. Transduced HSCs can be positively selected by low-dose O6BG/BCNU-treatment to achieve ~90% marking rates in peripheral blood. Initial proof-of-concept in murine models as well as in rhesus macaques demonstrates high level of g-globin expression after gene addition by a Sleeping Beauty transposase. While the current mobilization regimen-4 days of G-CSF injection followed by an injection of AMD3100/plerixafor on day 5-mobilizes HSCs from the bone marrow to the periphery, several issues exist. Despite widespread use as a mobilization agent in oncology, G-CSF is contra-indicated in patients with sickle cell disease. Additionally, G-CSF results in unselective bone marrow cell mobilization, which leads to leukocytosis and elevated numbers of cytokine-producing cells in the periphery that come into contact with HDAd particles, leading to high cytokine levels. Mobilized (committed) bone marrow cells in the periphery also sequester HDAd thus reducing the effective dose for primitive HSCs. Further, the five-day treatment regimen and high costs associated with G-CSF + plerixafor justify the development of an alternative mobilization regimen. A single-day, G-CSF-free mobilization regimen that mobilizes a high proportion of HSCs may therefore be preferred for in vivo gene therapy. Results . Here we tested HSC mobilization by truncated MGTA-145, a CXCR2 agonist, and plerixafor in the context of in vivo HSC transduction. CD46-transgenic animals were mobilized with GCSF + plerixafor (5 days) or with MGTA-145 + plerixafor (same-day treatment) and then injected one hour later with an integrating HDAd5/35++ mgmt/GFP vector. MGTA-145 + plerixafor resulted in robust mobilization of HSCs, less leukocytosis and no significant elevation of cytokines, as observed with G-CSF + plerixafor. With both mobilization regimens, after in vivo selection with O6BG/BCNU, >90% of PBMCs expressed GFP and marking rates were stable long-term. Mice were sacrificed 12 weeks after in vivo transduction and bone marrow lineage-negative cells were harvested for transplantation into secondary recipients. Stable transgene expression (>90% at week 16 after transplantation) was observed with both mobilization regimen in secondary recipients and multilineage engraftment was observed with MGTA-145 + plerixafor in primary and secondary recipients. Importantly, the mobilization with MGTA-145 + plerixafor worked efficiently in a mouse disease model for thalassemia (Hbbth3/CD46+/+). In this model, after in vivo transduction with an integrating HDAd5/35++mgmt/gamma-globin vector and in vivo selection, over 95% of peripheral red blood cells (RBCs) expressed human gamma-globin. The gamma-globin protein level reached 36% over mouse beta-globin. Phenotypic analyses showed a complete correction reflected by normal RBC morphology and absence of blood reticulocytosis, extramedullary hemopoiesis and hemosiderin deposition in spleen and liver sections. In secondary recipients of Lin- cells (harvested at week 14 from in vivo transduced Hbbth3/CD46+/+ mice), gamma-globin marking in RBCs was stable at 99% (currently at week 11 after transplantation). This demonstrates that MGTA145 + plerixafor mobilizes long-term repopulating HSCs. Conclusions . These data demonstrate that the combination of MGTA-145 and plerixafor could serve as an efficient and potentially safer one-day mobilization regimen for in vivo HSC gene therapy in patients with hemoglobinopathies. Disclosures Goncalves: Magenta Therapeutics: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Davis:Magenta Therapeutics: Current Employment, Current equity holder in publicly-traded company. Kiem:Rocket Pharma: Membership on an entity's Board of Directors or advisory committees; Umoja: Membership on an entity's Board of Directors or advisory committees; CSL: Consultancy; Homology Medicines: Membership on an entity's Board of Directors or advisory committees; Vor Biopharma: Membership on an entity's Board of Directors or advisory committees; Enochian: Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics: Consultancy. Lieber:Ensoma, Inc: Consultancy, Research Funding.

Curative in vivo hematopoietic stem cell gene therapy of murine thalassemia using large regulatory elements.

Recently, we demonstrated that hematopoietic stem/progenitor cell (HSPC) mobilization followed by intravenous injection of integrating, helper-dependent adenovirus HDAd5/35++ vectors resulted in efficient transduction of long-term repopulating cells and disease amelioration in mouse models after in vivo selection of transduced HSPCs. Acute innate toxicity associated with HDAd5/35++ injection was controlled by appropriate prophylaxis, making this approach feasible for clinical translation. Our ultimate goal is to use this technically simple in vivo HSPC transduction approach for gene therapy of thalassemia major or sickle cell disease. A cure of these diseases requires high expression levels of the therapeutic protein (γ- or β-globin), which is difficult to achieve with lentivirus vectors because of their genome size limitation not allowing larger regulatory elements to be accommodated. Here, we capitalized on the 35 kb insert capacity of HDAd5/35++ vectors to demonstrate that transcriptional regulatory regions of the β-globin locus with a total length of 29 kb can efficiently be transferred into HSPCs. The in vivo HSPC transduction resulted in stable γ-globin levels in erythroid cells that conferred a complete cure of murine thalassemia intermedia. Notably, this was achieved with a minimal in vivo HSPC selection regimen.

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.

The rat homolog to FX201, a gene therapy in development for the treatment of osteoarthritis, demonstrates dose-dependent decreases in the severity of cartilage and bone lesions following anterior cruciate ligament transection

Purpose: Acute injury to the anterior cruciate ligament (ACL) is a common cause of posttraumatic osteoarthritis (OA) in humans, and ACL transection (ACLT) in rats is an established animal model of traumatic injury-induced OA. FX201 is a novel interleukin-1 receptor antagonist (IL-1Ra) gene therapy for intra-articular (IA) administration. This helper-dependent adenovirus (HDAd)-based candidate is designed to induce the production of IL-1Ra in the presence of inflammation (via a nuclear factor-kappa B-responsive promoter) and is in development as a potential therapeutic agent for OA.

Chronic liver disease and impaired hepatic glycogen metabolism in argininosuccinate lyase deficiency.

BACKGROUNDLiver disease in urea cycle disorders (UCDs) ranges from hepatomegaly and chronic hepatocellular injury to cirrhosis and end-stage liver disease. However, the prevalence and underlying mechanisms are unclear.METHODSWe estimated the prevalence of chronic hepatocellular injury in UCDs using data from a multicenter, longitudinal, natural history study. We also used ultrasound with shear wave elastography and FibroTest to evaluate liver stiffness and markers of fibrosis in individuals with argininosuccinate lyase deficiency (ASLD), a disorder with high prevalence of elevated serum alanine aminotransferase (ALT). To understand the human observations, we evaluated the hepatic phenotype of the AslNeo/Neo mouse model of ASLD.RESULTSWe demonstrate a high prevalence of elevated ALT in ASLD (37%). Hyperammonemia and use of nitrogen-scavenging agents, 2 markers of disease severity, were significantly (P < 0.001 and P = 0.001, respectively) associated with elevated ALT in ASLD. In addition, ultrasound with shear wave elastography and FibroTest revealed increased echogenicity and liver stiffness, even in individuals with ASLD and normal aminotransferases. The AslNeo/Neo mice mimic the human disorder with hepatomegaly, elevated aminotransferases, and excessive hepatic glycogen noted before death (3-5 weeks of age). This excessive hepatic glycogen is associated with impaired hepatic glycogenolysis and decreased glycogen phosphorylase and is rescued with helper-dependent adenovirus expressing Asl using a liver-specific (ApoE) promoter.CONCLUSIONOur results link urea cycle dysfunction and impaired hepatic glucose metabolism and identify a mouse model of liver disease in the setting of urea cycle dysfunction.TRIAL REGISTRATIONThis study has been registered at ClinicalTrials.gov (NCT03721367, NCT00237315).FUNDINGFunding was provided by NIH, Burroughs Wellcome Fund, NUCDF, Genzyme/ACMG Foundation, and CPRIT.

A Single “All-in-One” Helper-Dependent Adenovirus to Deliver Donor DNA and CRISPR/Cas9 for Efficient Homology-Directed Repair

In this study, we developed a single helper-dependent adenovirus (HDAd) to deliver all of the components (donor DNA, CRISPR-associated protein 9 [Cas9], and guide RNA [gRNA]) needed to achieve high-efficiency gene targeting and homology-directed repair in transduced cells. We show that these “all-in-one” HDAds are up to 117-fold more efficient at gene targeting than donor HDAds that do not express CRISPR/Cas9 in human induced pluripotent stem cells (iPSCs). The vast majority (>90%) of targeted recombinants had only one allele targeted, and this was accompanied by high-frequency indel formation in the non-targeted allele at the site of Cas9 cleavage. These indels varied in size and nature, and included large deletions of ∼8 kb. The remaining minority of recombinants had both alleles targeted (so-called bi-allelic targeting). These all-in-one HDAds represent an important platform for accomplishing and expanding the utility of homology-directed repair, especially for difficult-to-transfect cells and for in vivo applications.

Exosome-Mediated Transfer of Anti-miR-33a-5p from Transduced Endothelial Cells Enhances Macrophage and Vascular Smooth Muscle Cell Cholesterol Efflux.

Atherosclerosis is a disease of large- and medium-sized arteries that is caused by cholesterol accumulation in arterial intimal cells, including macrophages and smooth muscle cells (SMC). Cholesterol accumulation in these cells can be prevented or reversed in preclinical models-and atherosclerosis reduced-by transgenesis that increases expression of molecules that control cholesterol efflux, including apolipoprotein AI (apoAI) and ATP-binding cassette subfamily A, member 1 (ABCA1). In a previous work, we showed that transduction of arterial endothelial cells (EC)-with a helper-dependent adenovirus (HDAd) expressing apoAI-enhanced EC cholesterol efflux in vitro and decreased atherosclerosis in vivo. Similarly, overexpression of ABCA1 in cultured EC increased cholesterol efflux and decreased inflammatory gene expression. These EC-targeted gene-therapy strategies might be improved by concurrent upregulation of cholesterol-efflux pathways in other intimal cell types. Here, we report modification of this strategy to enable delivery of therapeutic nucleic acids to cells of the sub-endothelium. We constructed an HDAd (HDAdXMoAntimiR33a5p) that expresses an antagomiR directed at miR-33a-5p (a microRNA that suppresses cholesterol efflux by silencing ABCA1). HDAdXMoAntimiR33a5p contains a sequence motif that enhances uptake of anti-miR-33a-5p into exosomes. Cultured EC release exosomes containing small RNA, including miR-33a-5p. After transduction with HDAdXMoAntimiR33a5p, EC-derived exosomes containing anti-miR-33a-5p accumulate in conditioned medium (CM). When this CM is added to macrophages or SMC, anti-miR-33a-5p is detected in these target cells. Exosome-mediated transfer of anti-miR-33a-5p reduces miR-33a-5p by ∼65-80%, increases ABCA1 protein by 1.6-2.2-fold, and increases apoAI-mediated cholesterol efflux by 1.4-1.6-fold (all p ≤ 0.01). These effects were absent in macrophages and SMC incubated in exosome-depleted CM. EC transduced with HDAdXMoAntimiR33a5p release exosomes that can transfer anti-miR-33a-5p to other intimal cell types, upregulating cholesterol efflux from these cells. This strategy provides a platform for genetic modification of intimal and medial cells, using a vector that transduces only EC.

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.

Abstract DP-003: TOWARD A HEMATOPOIETIC STEM CELL-BASED PROPHYLACTIC IMMUNO-GENE THERAPY APPROACH FOR OVARIAN CANCER

Abstract Our ultimate goal is to develop a long-lasting, cost-efficient, and technically simple approach that allows for the immuno-prophylaxis of cancer in patients with high-risk for disease recurrence and, ultimately, in patients with cancer-predisposing inherited mutations. Our approach is based on in vivo genetic modification of hematopoietic stem cells (HSCs). Because HSCs are self-renewing and give rise to all blood cell lineages, they provide a life-long source of transgene modified myeloid and lymphoid cells that, during tumor development, infiltrate the tumor and support tumor growth. Our in vivo HSC transduction approach involves subcutaneous 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 (HD-Ad5/35++) vector system. In preliminary studies we used an integrating HDAd5/35++ vector expressing GFP and mgtmP140K under the ubiquitously active EF1a promoter. We demonstrated in adequate mouse models that HSCs transduced in the periphery home back to the bone marrow where they persist long-term. Transgene expression in peripheral blood cells can be achieved by O6BG/BCNU injection. Short-term exposure of in vivo transduced mice to low O6BG/BCNU doses resulted in stable GFP expression in 80% of peripheral blood cells and, in mice with implanted syngeneic tumors, in 80% of tumor infiltrating leukocytes. The predominant GFP-positive cell type in two syngeneic tumor models was tumor-associated neutrophils (TANs) (Ly6G+) and macrophages (TAMs) (F4/80+/MHCII+). GFP+ TANs and TAMs were detectable at very early stage of tumor development. To avoid adverse reactions using our in vivo HSC transduction/selection approach, the expression of therapeutic transgenes has to i) be localized to the tumor, ii) be automatically activated only when the tumor begins to develop, and iii) cease when the tumor disappears. To develop such an expression system, we determined (by miRNA-Seq and miRNA-array) the micro-RNA profile in GFP+ cells isolated from the bone marrow HSCs, spleen, PBMCs, and tumors of in vivo transduced mice. We found four miRNAs that were expressed at high levels in HSCs, splenocytes and PBMCs, but were absent in tumor-associated leukocytes. By inserting four copies of the corresponding target sites into the 3'UTR of our GFP transgene, the corresponding mRNA is degraded in all cells except tumor-associated leukocytes allowing for tumor-restricted transgene expression. Studies on validating this system using GFP as a reporter gene are nearly completed. Furthermore, we have generated HDAd5/35++ vectors expressing scFv specific to mouse CTLA4 or PDL1 under miRNA control. We have performed in vivo HSC transduction-O6BG/BCNU treatment with these vectors and will implant syngeneic tumors at the beginning of August. We expect that intratumoral anti-CTLA4 or anti-PDL1 expression from TANs/TAMs at early stages of tumor development will overcome the immunosuppressive tumor environment allowing effector T-cells to stop tumor growth. The outcome of these studies will be presented. We have also started testing the prophylactic efficacy of our approach in oncogene-transgenic mice that develop spontaneous tumors. Citation Format: Chang Li, Paul Valdmanis, Andre Lieber. TOWARD A HEMATOPOIETIC STEM CELL-BASED PROPHYLACTIC IMMUNO-GENE THERAPY APPROACH FOR OVARIAN CANCER [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr DP-003.

Bi-allelic Homology-Directed Repair with Helper-Dependent Adenoviruses

We describe a strategy to achieve footprintless bi-allelic homology-directed repair (HDR) using helper-dependent adenoviruses (HDAds). This approach utilizes two HDAds to deliver the donor DNA. These two HDAds are identical except for their selectable marker. One expresses the puromycin N-acetyltransferase-herpes simplex virus I thymidine kinase fusion gene (PACTk), while the other expresses the hygromycin phosphotransferase-herpes simplex virus I thymidine kinase fusion gene (HyTk). Therefore, puromycin and hygromycin double resistance can be used to select for targeted HDAd integration into both alleles. Subsequently, piggyBac-mediated excision of both PACTk and HyTk will confer resistance to gancyclovir, resulting in footprintless HDR at both alleles. However, gene-targeting frequency was not high enough to achieve simultaneous targeting at both alleles. Instead, sequential targeting, whereby the two alleles were targeted one at a time, was required in order to achieve bi-allelic HDR with HDAd.

High-level protein production in erythroid cells derived from in vivo transduced hematopoietic stem cells

AbstractWe developed an in vivo hematopoietic stem cell (HSC) transduction approach that involves HSC mobilization from the bone marrow into the peripheral bloodstream and the IV injection of an integrating, helper-dependent adenovirus (HDAd5/35++) vector system. HDAd5/35++ vectors target human CD46, a receptor that is abundantly expressed on primitive HSCs. Transgene integration is achieved by a hyperactive Sleeping Beauty transposase (SB100x) and transgene marking in peripheral blood cells can be increased by in vivo selection. Here we directed transgene expression to HSC-derived erythroid cells using β-globin regulatory elements. We hypothesized that the abundance and systemic distribution of erythroid cells can be harnessed for high-level production of therapeutic proteins. We first demonstrated that our approach allowed for sustained, erythroid-lineage specific GFP expression and accumulation of GFP protein in erythrocytes. Furthermore, after in vivo HSC transduction/selection in hCD46-transgenic mice, we demonstrated stable supraphysiological plasma concentrations of a bioengineered human factor VIII, termed ET3. High-level ET3 production in erythroid cells did not affect erythropoiesis. A phenotypic correction of bleeding was observed after in vivo HSC transduction of hCD46+/+/F8−/− hemophilia A mice despite high plasma anti-ET3 antibody titers. This suggests that ET3 levels were high enough to provide sufficient noninhibited ET3 systemically and/or locally (in blood clots) to control bleeding. In addition to its relevance for hemophilia A gene therapy, our approach has implications for the therapy of other inherited or acquired diseases that require high levels of therapeutic proteins in the blood circulation.

No more helper adenovirus: production of gutless adenovirus (GLAd) free of adenovirus and replication-competent adenovirus (RCA) contaminants

Gene therapy is emerging as an effective treatment option for various inherited genetic diseases. Gutless adenovirus (GLAd), also known as helper-dependent adenovirus (HDAd), has many notable characteristics as a gene delivery vector for this particular type of gene therapy, including broad tropism, high infectivity, a large transgene cargo capacity, and an absence of integration into the host genome. Additionally, GLAd ensures long-term transgene expression in host organisms owing to its minimal immunogenicity, since it was constructed following the deletion of all the genes from an adenovirus. However, the clinical use of GLAd for the treatment of inherited genetic diseases has been hampered by unavoidable contamination of the highly immunogenic adenovirus used as a helper for GLAd production. Here, we report the production of GLAd in the absence of a helper adenovirus, which was achieved with a helper plasmid instead. Utilizing this helper plasmid, we successfully produced large quantities of recombinant GLAd. Importantly, our helper plasmid-based system exclusively produced recombinant GLAd with no generation of helper plasmid-originating adenovirus and replication-competent adenovirus (RCA). The recombinant GLAd that was produced efficiently delivered transgenes regardless of their size and exhibited therapeutic potential for Huntington’s disease (HD) and Duchenne muscular dystrophy (DMD). Our data indicate that our helper plasmid-based GLAd production system could become a new platform for GLAd-based gene therapy.

Helper-Dependent Adenovirus Transduces the Human and Rat Retina but Elicits an Inflammatory Reaction When Delivered Subretinally in Rats.

The identification of >100 genes causing inherited retinal degeneration and the promising results of recent gene augmentation trials have led to an increase in the number of studies investigating the preclinical efficacy of viral-mediated gene transfer. Despite success using adeno-associated viruses, many disease-causing genes, such as ABCA4 or USH2A, are too large to fit into these vectors. One option for large gene delivery is the family of integration-deficient helper-dependent adenoviruses (HDAds), which efficiently transduce postmitotic neurons. However, HDAds have been shown in other organ systems to elicit an immune response, and the immunogenicity of HDAds in the retina has not been characterized. In this study, HDAd serotype 5 (HDAd5) was found to successfully transduce rod and cone photoreceptors in ex vivo human retinal organ cultures. The ocular inflammatory response to subretinal injection of the HDAd5 was evaluated using a rat model. Subretinal injection of HDAd5 carrying cytomegalovirus promoter-driven enhanced green fluorescent protein (HDAd5-CMVp-eGFP) elicited a robust inflammatory response by 3 days postinjection. This reaction included vitreous infiltration of ionized calcium-binding adapter molecule 1 (Iba1)-positive monocytes and increased expression of the proinflammatory protein, intercellular adhesion molecule 1 (ICAM-1). By 7 days postinjection, most Iba1-positive infiltrates migrated into the neural retina and ICAM-1 expression was significantly increased compared with buffer-injected control eyes. At 14 days postinjection, Iba1-positive cells persisted in the retinas of HDAd5-injected eyes, and there was thinning of the outer nuclear layer. Subretinal injection of an empty HDAd5 virus was used to confirm that the inflammatory response was in response to the HDAd5 vector and not due to eGFP-induced overexpression cytotoxicity. Subretinal injection of lower doses of HDAd5 dampened the inflammatory response, but also eGFP expression. Despite their larger carrying capacity, further work is needed to elucidate the inflammatory pathways involved and to identify an immunomodulation paradigm sufficient for safe and effective transfer of large genes to the retina using HDAd5.

In Vitro Validation of a CRISPR-Mediated CFTR Correction Strategy for Preclinical Translation in Pigs.

Early efforts in cystic fibrosis (CF) gene therapy faced major challenges in delivery efficiency and sustained therapeutic gene expression. Recent advancements in engineered site-specific endonucleases such as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 make permanent CF transmembrane conductance regulator (CFTR) gene correction possible. However, because of safety concerns of the CRISPR/Cas9 system and challenges in in vivo delivery to inflamed CF airway, CRISPR-based gene correction strategies need to be tested in proper animal models. In this study, we aimed at creating vectors for testing CFTR gene correction in pig models. We constructed helper-dependent adenoviral (HD-Ad) vectors to deliver CRISPR/Cas9 and a donor template (a 6 kb LacZ or 8.7 kb human CFTR expression cassette) into cultured pig cells. We demonstrated precise integration of each donor into the GGTA1 safe harbor through Cas9-induced homology directed repair with 3 kb homology arms. In addition, we showed that both LacZ and hCFTR were persistently expressed in transduced cells. Furthermore, we created a CFTR-deficient cell line for testing CFTR correction. We detected hCFTR mRNA and protein expression in cells transduced with the hCFTR vector. We also demonstrated CFTR function in the CF cells transduced with the HD-Ad delivering the CRISPR-Cas9 system and hCFTR donor at late cellular passages using the membrane potential sensitive dye-based assay (FLIPR®). Combined with our previous report on gene delivery to pig airway basal cells, these data provide the feasibility of testing CRISPR/Cas9-mediated permanent human CFTR correction through HD-Ad vector delivery in pigs.

Production of CRISPR/Cas9-Mediated Self-Cleaving Helper-Dependent Adenoviruses

Prolonged expression of CRISPR/Cas9 raises concerns about off-target cleavage, cytotoxicity, and immune responses. To address these issues, we have developed a system to produce helper-dependent adenoviruses that express CRISPR/Cas9 to direct cleavage of the vectors’ own genome after transduction of target cells. To prevent self-cleavage during vector production, it was necessary to downregulate Cas9 mRNA as well as inhibit Cas9 protein activity. Cas9 mRNA downregulation was achieved by inserting the target sequences for the helper-virus-encoded miRNA, mivaRNAI, and producer-cell-encoded miRNAs, hsa-miR183-5p, and hsa-miR218-5p, into the 3′ UTR of the HDAd-encoded Cas9 expression cassette. Cas9 protein activity was inhibited by expressing anti-CRISPR proteins AcrIIA2 and AcrAII4 from both the producer cells and the helper virus. After purification, these helper-dependent adenoviruses will perform CRISPR/Cas9-mediated self-cleavage in the transduced target cells, thereby limiting the duration of Cas9 expression and thus represent an important platform for improving the safety of gene editing by CRISPR/Cas9.

Hepatic glutamine synthetase augmentation enhances ammonia detoxification.

The urea cycle and glutamine synthetase (GS) are the two main pathways for waste nitrogen removal and their deficiency results in hyperammonemia. Here, we investigated the efficacy of liver-specific GS overexpression for therapy of hyperammonemia. To achieve hepatic GS overexpression, we generated a helper-dependent adenoviral (HDAd) vector expressing the murine GS under the control of a liver-specific expression cassette (HDAd-GS). Compared to mice injected with a control vector expressing an unrelated reporter gene (HDAd-alpha-fetoprotein), wild-type mice with increased hepatic GS showed reduced blood ammonia levels and a concomitant increase of blood glutamine after intraperitoneal injections of ammonium chloride, whereas blood urea was unaffected. Moreover, injection of HDAd-GS reduced blood ammonia levels at baseline and protected against acute hyperammonemia following ammonia challenge in a mouse model with conditional hepatic deficiency of carbamoyl phosphate synthetase 1 (Cps1), the initial and rate-limiting step of ureagenesis. In summary, we found that upregulation of hepatic GS reduced hyperammonemia in wild-type and Cps1-deficient mice, thus confirming a key role of GS in ammonia detoxification. These results suggest that hepatic GS augmentation therapy has potential for treatment of both primary and secondary forms of hyperammonemia.