In vivo HSC prime editing rescues sickle cell disease in a mouse model.

Sickle cell disease (SCD) is a monogenic disease caused by a nucleotide mutation in the ß-globin gene. Current gene therapy studies are mainly focused on lentiviral vector-mediated gene addition or CRISPR/Cas9-mediated fetal globin reactivation, leaving the root cause unfixed. We developed a vectorized prime editing system that can directly repair the SCD mutation in hematopoietic stem cells (HSCs) in vivo in a SCD mouse model (CD46/Townes mice). Our approach involved a single intravenous injection of a nonintegrating, prime editor-expressing viral vector into mobilized CD46/Townes mice and low-dose drug selection in vivo. This procedure resulted in the correction of ∼40% of ßS alleles in HSCs. On average, 43% of sickle hemoglobin was replaced by adult hemoglobin, thereby greatly mitigating the SCD phenotypes. Transplantation in secondary recipients demonstrated that long-term repopulating HSCs were edited. Highly efficient target site editing was achieved with minimal generation of insertions and deletions and no detectable off-target editing. Because of its simplicity and portability, our in vivo prime editing approach has the potential for application in resource-poor countries where SCD is prevalent.

Stable HIV decoy receptor expression after in vivo HSC transduction in mice and NHPs: Safety and efficacy in protection from SHIV.

We aim to develop an in vivo hematopoietic stem cell (HSC) gene therapy approach for persistent control/protection of HIV-1 infection based on the stable expression of a secreted decoy protein for HIV receptors CD4 and CCR5 (eCD4-Ig) from blood cells. HSCs in mice and a rhesus macaque were mobilized from the bone marrow and transduced by an intravenous injection of HSC-tropic, integrating HDAd5/35++ vectors expressing rhesus eCD4-Ig. In vivo HSC transduction/selection resulted in stable serum eCD4-Ig levels of ∼100 µg/mL (mice) and >20 µg/mL (rhesus) with half maximal inhibitory concentrations (IC50s) of 1 µg/mL measured by an HIV neutralization assay. After simian-human-immunodeficiency virus D (SHIV.D) challenge of rhesus macaques injected with HDAd-eCD4-Ig or a control HDAd5/35++ vector, peak plasma viral load levels were ∼50-fold lower in the eCD4-Ig-expressing animal. Furthermore, the viral load was lower in tissues with the highest eCD4-Ig expression, specifically the spleen and lymph nodes. SHIV.D challenge triggered a selective expansion of transduced CD4+CCR5+ cells, thereby increasing serum eCD4-Ig levels. The latter, however, broke immune tolerance and triggered anti-eCD4-Ig antibody responses, which could have contributed to the inability to eliminate SHIV.D. Our data will guide us in the improvement of the in vivo approach. Clearly, our conclusions need to be validated in larger animal cohorts.

In Vivo HSC Gene Therapy Using a Bi-modular HDAd5/35++ Vector Cures Sickle Cell Disease in a Mouse Model.

We have recently reported that, after in vivo hematopoietic stem cell/progenitor (HSPC) transduction with HDAd5/35++ vectors, SB100x transposase-mediated γ-globin gene addition achieved 10%-15% γ-globin of adult mouse globin, resulting in significant but incomplete phenotypic correction in a thalassemia intermedia mouse model. Furthermore, genome editing of a γ-globin repressor binding site within the γ-globin promoter by CRISPR-Cas9 results in efficient reactivation of endogenous γ-globin. Here, we aimed to combine these two mechanisms to obtain curative levels of γ-globin after in vivo HSPC transduction. We generated a HDAd5/35++ adenovirus vector (HDAd-combo) containing both modules and tested it in vitro and after in vivo HSPC transduction in healthy CD46/ß-YAC mice and in a sickle cell disease mouse model (CD46/Townes). Compared to HDAd vectors containing either the γ-globin addition or the CRISPR-Cas9 reactivation units alone, in vivo HSC transduction of CD46/Townes mice with the HDAd-combo resulted in significantly higher γ-globin in red blood cells, reaching 30% of that of adult human α and ßS chains and a complete phenotypic correction of sickle cell disease.

Reactivation of γ-globin in adult ß-YAC mice after ex vivo and in vivo hematopoietic stem cell genome editing.

Disorders involving ß-globin gene mutations, primarily ß-thalassemia and sickle cell disease, represent a major target for hematopoietic stem/progenitor cell (HSPC) gene therapy. This includes CRISPR/Cas9-mediated genome editing approaches in adult CD34+ cells aimed toward the reactivation of fetal γ-globin expression in red blood cells. Because models involving erythroid differentiation of CD34+ cells have limitations in assessing γ-globin reactivation, we focused on human ß-globin locus-transgenic (ß-YAC) mice. We used a helper-dependent human CD46-targeting adenovirus vector expressing CRISPR/Cas9 (HDAd-HBG-CRISPR) to disrupt a repressor binding region within the γ-globin promoter. We transduced HSPCs from ß-YAC/human CD46-transgenic mice ex vivo and subsequently transplanted them into irradiated recipients. Furthermore, we used an in vivo HSPC transduction approach that involves HSPC mobilization and the intravenous injection of HDAd-HBG-CRISPR into ß-YAC/CD46-transgenic mice. In both models, we demonstrated efficient target site disruption, resulting in a pronounced switch from human ß- to γ-globin expression in red blood cells of adult mice that was maintained after secondary transplantation of HSPCs. In long-term follow-up studies, we did not detect hematological abnormalities, indicating that HBG promoter editing does not negatively affect hematopoiesis. This is the first study that shows successful in vivo HSPC genome editing by CRISPR/Cas9.

Targeted Integration and High-Level Transgene Expression in AAVS1 Transgenic Mice after In Vivo HSC Transduction with HDAd5/35++ Vectors.

Our goal is the development of in vivo hematopoietic stem cell (HSC) transduction technology with targeted integration. To achieve this, we modified helper-dependent HDAd5/35++ vectors to express a CRISPR/Cas9 specific to the "safe harbor" adeno-associated virus integration site 1 (AAVS1) locus and to provide a donor template for targeted integration through homology-dependent repair. We tested the HDAd-CRISPR + HDAd-donor vector system in AAVS1 transgenic mice using a standard ex vivo HSC gene therapy approach as well as a new in vivo HSC transduction approach that involves HSC mobilization and intravenous HDAd5/35++ injections. In both settings, the majority of treated mice had transgenes (GFP or human γ-globin) integrated into the AAVS1 locus. On average, >60% of peripheral blood cells expressed the transgene after in vivo selection with low-dose O6BG/bis-chloroethylnitrosourea (BCNU). Ex vivo and in vivo HSC transduction and selection studies with HDAd-CRISPR + HDAd-globin-donor resulted in stable γ-globin expression at levels that were significantly higher (>20% γ-globin of adult mouse globin) than those achieved in previous studies with a SB100x-transposase-based HDAd5/35++ system that mediates random integration. The ability to achieve therapeutically relevant transgene expression levels after in vivo HSC transduction and selection and targeted integration make our HDAd5/35++-based vector system a new tool in HSC gene therapy.

Studies on the Interaction of Tumor-Derived HD5 Alpha Defensins with Adenoviruses and Implications for Oncolytic Adenovirus Therapy.

Defensins are small antimicrobial peptides capable of neutralizing human adenovirus (HAdV) in vitro by binding capsid proteins and blocking endosomal escape of virus. In humans, the alpha defensin HD5 is produced by specialized epithelial cells of the gastrointestinal and genito-urinary tracts. Here, we demonstrate, using patient biopsy specimens, that HD5 is also expressed as an active, secreted peptide by epithelial ovarian and lung cancer cells in situ This finding prompted us to study the role of HD5 in infection and spread of replication-competent, oncolytic HAdV type 3 (HAdV3). HAdV3 produces large amounts of penton-dodecahedra (PtDd), virus-like particles, during replication. We have previously shown that PtDd are involved in opening epithelial junctions, thus facilitating lateral spread of de novo-produced virions. Here, we describe a second function of PtDd, namely, the blocking of HD5. A central tool to prove that viral PtDd neutralize HD5 and support spread of progeny virus was an HAdV3 mutant virus in which formation of PtDd was disabled (mut-Ad3GFP, where GFP is green fluorescent protein). We demonstrated that viral spread of mut-Ad3GFP was blocked by synthetic HD5 whereas that of the wild-type (wt) form (wt-Ad3GFP) was only minimally impacted. In human colon cancer Caco-2 cells, induction of cellular HD5 expression by fibroblast growth factor 9 (FGF9) significantly inhibited viral spread and progeny virus production of mut-Ad3GFP but not of wt-Ad3GFP. Finally, the ectopic expression of HD5 in tumor cells diminished the in vivo oncolytic activity of mut-Ad3GFP but not of wt-Ad3GFP. These data suggest a new mechanism of HAdV3 to overcome innate antiviral host responses. Our study has implications for oncolytic adenovirus therapy.IMPORTANCE Previously, it has been reported that human defensin HD5 inactivates specific human adenoviruses by binding to capsid proteins and blocking endosomal escape of virus. The central new findings described in our manuscript are the following: (i) the discovery of a new mechanism used by human adenovirus serotype 3 to overcome innate antiviral host responses that is based on the capacity of HAdV3 to produce subviral penton-dodecahedral particles that act as decoys for HD5, thus preventing the inactivation of virus progeny produced upon replication; (ii) the demonstration that ectopic HD5 expression in cancer cells decreases the oncolytic efficacy of a serotype 5-based adenovirus vector; and (iii) the demonstration that epithelial ovarian and lung cancers express HD5. The study improves our understanding of how adenoviruses establish infection in epithelial tissues and has implications for cancer therapy with oncolytic adenoviruses.

CYR61 (CCN1) overexpression induces lung injury in mice.

Cysteine-rich protein-61 (CYR61), also known as connective tissue growth factor, CYR61, and nephroblastoma overexpressed gene 1 (CCN1), is a heparin-binding protein member of the CCN family of matricellular proteins. Gene expression profiles showed that Cyr61 is upregulated in human acute lung injury (ALI), but its functional role is unclear. We hypothesized that CYR61 contributes to ALI in mice. First, we demonstrated that CYR61 expression increases after bleomycin-induced lung injury. We then used adenovirus-mediated gene transfer to determine whether CYR61 overexpression in the lungs was sufficient to cause ALI. Mice instilled with CYR61 adenovirus showed greater weight loss, increased bronchoalveolar lavage total neutrophil counts, increased protein concentrations, and increased mortality compared with mice instilled with empty-vector adenovirus. Immunohistochemical studies in lungs from humans with idiopathic pulmonary fibrosis revealed CYR61 expression on the luminal membrane of alveolar epithelial cells in areas of injury. We conclude that CYR61 is upregulated in ALI and that CYR61 overexpression exacerbates ALI in mice.

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 in vivo 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 in vitro. 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 in vivo 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 in vivo 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 in vivo approach.

Fas-deficient mice have impaired alveolar neutrophil recruitment and decreased expression of anti-KC autoantibody:KC complexes in a model of acute lung injury.

BACKGROUND: Exposure to mechanical ventilation enhances lung injury in response to various stimuli, such as bacterial endotoxin (LPS). The Fas/FasL system is a receptor ligand system that has dual pro-apoptotic and pro-inflammatory functions and has been implicated in the pathogenesis of lung injury. In this study we test the hypothesis that a functioning Fas/FasL system is required for the development of lung injury in mechanically ventilated mice. METHODS: C57BL/6 (B6) and Fas-deficient lpr mice were exposed to either intra-tracheal PBS followed by spontaneous breathing or intra-tracheal LPS followed by four hours mechanical ventilation with tidal volumes of 10 mL/kg, respiratory rate of 150 breaths per minute, inspired oxygen 0.21 and positive end expiratory pressure (PEEP) of 3 cm of water. RESULTS: Compared with the B6 mice, the lpr mice showed attenuation of the neutrophilic response as measured by decreased numbers of BAL neutrophils and lung myeloperoxidase activity. Interestingly, the B6 and lpr mice had similar concentrations of pro-inflammatory cytokines, including CXCL1 (KC), and similar measurements of permeability and apoptosis. However, the B6 mice showed greater deposition of anti-KC:KC immune complexes in the lungs, as compared with the lpr mice. CONCLUSIONS: We conclude that a functioning Fas/FasL system is required for full neutrophilic response to LPS in mechanically ventilated mice.

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

We 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.

Translational development of a tumor junction opening technology.

Our goal is to overcome treatment resistance in ovarian cancer patients which occurs in most cases after an initial positive response to chemotherapy. A central resistance mechanism is the maintenance of desmoglein-2 (DSG2) positive tight junctions between malignant cells that prevents drug penetration into the tumor. We have generated JO4, a recombinant protein that binds to DSG2 resulting in the transient opening of junctions in epithelial tumors. Here we present studies toward the clinical translation of c-JO4 in combination with PEGylated liposomal doxorubicin/Doxil for ovarian cancer therapy. A manufacturing process for cGMP compliant production of JO4 was developed resulting in c-JO4. GLP toxicology studies using material from this process in DSG2 transgenic mice and cynomolgus macaques showed no treatment-related toxicities after intravenous injection at doses reaching 24 mg/kg. Multiple cycles of intravenous c-JO4 plus Doxil (four cycles, 4 weeks apart, simulating the treatment regimen in the clinical trial) elicited antibodies against c-JO4 that increased with each cycle and were accompanied by elevation of pro-inflammatory cytokines IL-6 and TNFα. Pretreatment with steroids and cyclophosphamide reduced anti-c-JO4 antibody response and blunted cytokine release. Our data indicate acceptable safety of our new treatment approach if immune reactions are monitored and counteracted with appropriate immune suppression.

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 ß-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.

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

[This corrects the article DOI: 10.1016/j.omtm.2021.12.003.].

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.

In vivo base editing by a single i.v. vector injection for treatment of hemoglobinopathies.

Individuals with ß-thalassemia or sickle cell disease and hereditary persistence of fetal hemoglobin (HPFH) possessing 30% fetal hemoglobin (HbF) appear to be symptom free. Here, we used a nonintegrating HDAd5/35++ vector expressing a highly efficient and accurate version of an adenine base editor (ABE8e) to install, in vivo, a -113 A>G HPFH mutation in the γ-globin promoters in healthy CD46/ß-YAC mice carrying the human ß-globin locus. Our in vivo hematopoietic stem cell (HSC) editing/selection strategy involves only s.c. and i.v. injections and does not require myeloablation and HSC transplantation. In vivo HSC base editing in CD46/ß-YAC mice resulted in > 60% -113 A>G conversion, with 30% γ-globin of ß-globin expressed in 70% of erythrocytes. Importantly, no off-target editing at sites predicted by CIRCLE-Seq or in silico was detected. Furthermore, no critical alterations in the transcriptome of in vivo edited mice were found by RNA-Seq. In vitro, in HSCs from ß-thalassemia and patients with sickle cell disease, transduction with the base editor vector mediated efficient -113 A>G conversion and reactivation of γ-globin expression with subsequent phenotypic correction of erythroid cells. Because our in vivo base editing strategy is safe and technically simple, it has the potential for clinical application in developing countries where hemoglobinopathies are prevalent.

In vivo HSPC gene therapy with base editors allows for efficient reactivation of fetal γ-globin in ß-YAC mice.

Base editors are capable of installing precise genomic alterations without creating double-strand DNA breaks. In this study, we targeted critical motifs regulating γ-globin reactivation with base editors delivered via HDAd5/35++ vectors. Through optimized design, we successfully produced a panel of cytidine and adenine base editor (ABE) vectors targeting the erythroid BCL11A enhancer or recreating naturally occurring hereditary persistence of fetal hemoglobin (HPFH) mutations in the HBG1/2 promoter. All 5 tested vectors efficiently installed target base conversion and led to γ-globin reactivation in human erythroid progenitor cells. We observed ~23% γ-globin protein production over ß-globin, when using an ABE vector (HDAd-ABE-sgHBG-2) specific to the -113A>G HPFH mutation. In a ß-YAC mouse model, in vivo hematopoietic progenitor/stem cell (HSPC) transduction with HDAd-ABE-sgHBG-2 followed by in vivo selection resulted in >40% γ-globin+ erythrocytes in the peripheral blood. This result corresponded to 21% γ-globin production over human ß-globin. The average -113A>G conversion in total bone marrow cells was 20%. No alterations in hematological parameters, erythropoiesis, and bone marrow cellular composition were observed after treatment. No detectable editing was found at top-scoring, off-target genomic sites. Bone marrow lineage-negative cells from primary mice were capable of reconstituting secondary transplant-recipient mice with stable γ-globin expression. Importantly, the advantage of base editing over CRISPR/Cas9 was reflected by the markedly lower rates of intergenic HBG1/2 deletion and the absence of detectable toxicity in human CD34+ cells. Our observations suggest that HDAd-vectorized base editors represent a promising strategy for precise in vivo genome engineering for the treatment of ß-hemoglobinopathies.

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-ß (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 ß-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.

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 ß-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 ß-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.

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

We 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 ß-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.