The glucocorticoid receptor elicited proliferative response in human erythropoiesis is BCL11A-dependent.

Prior evidence indicates that the erythroid cellular response to glucocorticoids (GC) has developmental specificity, namely, that developmentally more advanced cells that are undergoing or have undergone fetal to adult globin switching are more responsive to GC-induced expansion. To investigate the molecular underpinnings of this, we focused on the major developmental globin regulator BCL11A. We compared: a) levels of expression and nuclear content of BCL11A in adult erythroid cells upon GC stimulation; b) response to GC of CD34+ cells from patients with BCL11A microdeletions and reduced BCL11A expression, and; c) response to GC of two cellular models (HUDEP-2 and adult CD34+ cells) before and after reduction of BCL11A expression by shRNA. We observed that: a) GC-expanded erythroid cells from a large cohort of blood donors displayed amplified expression and nuclear accumulation of BCL11A; b) CD34+ cells from BCL11A microdeletion patients generated fewer erythroid cells when cultured with GC compared to their parents, while the erythroid expansion of the patients was similar to that of their parents in cultures without GC, and; c) adult CD34+ cells and HUDEP-2 cells with shRNA-depleted expression of BCL11A exhibit reduced expansion in response to GC. In addition, RNA-seq profiling of shRNA-BCL11A CD34+ cells cultured with and without GC was similar (very few differentially expressed genes), while GC-specific responses (differential expression of GILZ and of numerous additional genes) were observed only in controls cells with unperturbed BCL11A expression. These data indicate that BCL11A is an important participant of certain aspects of the stress pathway sustained by GC.

Patients with hypercortisolemic Cushing disease possess a distinct class of hematopoietic progenitor cells leading to erythrocytosis.

Although human cell cultures stimulated with dexamethasone suggest that the glucocorticoid receptor (GR) activates stress erythropoiesis, the effects of GR activation on erythropoiesis in vivo remain poorly understood. We characterized the phenotype of a large cohort of patients with Cushing disease, a rare condition associated with elevated cortisol levels. Results from hypercortisolemic patients with active Cushing disease were compared with those obtained from eucortisolemic patients after remission and from volunteers without the disease. Patients with active Cushing disease exhibited erythrocytosis associated with normal hemoglobin F levels. In addition, their blood contained elevated numbers of GR-induced CD163+ monocytes and a unique class of CD34+ cells expressing CD110, CD36, CD133 and the GR-target gene CXCR4. When cultured, these CD34+ cells generated similarly large numbers of immature erythroid cells in the presence and absence of dexamethasone, with raised expression of the GR-target gene GILZ. Of interest, blood from patients with Cushing disease in remission maintained high numbers of CD163+ monocytes and, although their CD34+ cells had a normal phenotype, these cells were unresponsive to added dexamethasone. Collectively, these results indicate that chronic exposure to excess glucocorticoids in vivo leads to erythrocytosis by generating erythroid progenitor cells with a constitutively active GR. Although remission rescues the erythrocytosis and the phenotype of the circulating CD34+ cells, a memory of other prior changes is maintained in remission.

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.].

CD4+c-Met+Itgα4+ T cell subset promotes murine neuroinflammation.

OBJECTIVE: c-Met, a tyrosine kinase receptor, is the unique receptor for hepatocyte growth factor (HGF). The HGF/c-Met axis is reported to modulate cell migration, maturation, cytokine production, and antigen presentation. Here, we report that CD4+c-Met+ T cells are detected at increased levels in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis (MS). METHODS: c-Met expression by CD4+ T cells was analyzed mostly by flow cytometry and by immunohistochemistry from mice and human PBMCs. The in vivo role of CD4+c-Met+ T cells was assessed in EAE. RESULTS: CD4+c-Met+ T cells found in the CNS during EAE peak disease are characterized by a pro-inflammatory phenotype skewed towards a Th1 and Th17 polarization, with enhanced adhesion and transmigration capacities correlating with increased expression of integrin α4 (Itgα4). The adoptive transfer of Itgα4-expressing CD4+Vα3.2+c-Met+ T cells induces increased disease severity compared to CD4+Vα3.2+c-Met- T cells. Finally, CD4+c-Met+ T cells are detected in the brain of MS patients, as well as in the blood with a higher level of Itgα4. These results highlight c-Met as an immune marker of highly pathogenic pro-inflammatory and pro-migratory CD4+ T lymphocytes associated with neuroinflammation.

α4-Integrin deficiency in human CD34+ cells engenders precocious erythroid differentiation but inhibits enucleation.

The functional impact of integrin expression in erythropoiesis has been previously emphasized through its decisive influence on erythroid cell-microenvironment (matrix and cellular) interactions, especially under conditions of stress. Beyond that, in several in vitro studies the relationship between the two erythroid integrins, α4 and α5, has been incongruous in terms of a proliferative support, either synergistic or antagonistic, whereas a dominant influence of α4 integrin on terminal erythropoiesis in vitro and in vivo has been consistently emphasized. However, the specific cellular and molecular details of this effect have not been defined, especially for human cells. In the study described here, we cultured human CD34+ progenitor cells with induced deficiency of α4 integrin (shRNAα4) under erythroid differentiation conditions, in which expanded erythroid progenitor cells are directed to terminal erythroid maturation stages in the absence of any microenvironmental influence. Our data indicate that early proliferative expansion in cells lacking α4 expression is significantly limited, but although erythroid cell differentiation can proceed normally to terminal stages, their enucleation is drastically impaired. This novel aspect of α4 integrin participation in the enucleation process in vitro resonates on the lack of in vivo enucleation of primitive erythroid cells lacking any integrin expression but affecting adult cells only under stress conditions.

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.

Investigating the Barrier Activity of Novel, Human Enhancer-Blocking Chromatin Insulators for Hematopoietic Stem Cell Gene Therapy.

Despite the unequivocal success of hematopoietic stem and progenitor cell gene therapy, limitations still exist including genotoxicity and variegation/silencing of transgene expression. A class of DNA regulatory elements known as chromatin insulators (CIs) can mitigate both vector transcriptional silencing (barrier CIs) and vector-induced genotoxicity (enhancer-blocking CIs) and have been proposed as genetic modulators to minimize unwanted vector/genome interactions. Recently, a number of human, small-sized, and compact CIs bearing strong enhancer-blocking activity were identified. To ultimately uncover an ideal CI with a dual, enhancer-blocking and barrier activity, we interrogated these elements in vitro and in vivo. After initial screening of a series of these enhancer-blocking insulators for potential barrier activity, we identified three distinct categories with no, partial, or full protection against transgene silencing. Subsequently, the two CIs with full barrier activity (B4 and C1) were tested for their ability to protect against position effects in primary cells, after incorporation into lentiviral vectors (LVs) and transduction of human CD34+ cells. B4 and C1 did not adversely affect vector titers due to their small size, while they performed as strong barrier insulators in CD34+ cells, both in vitro and in vivo, shielding transgene's long-term expression, more robustly when placed in the forward orientation. Overall, the incorporation of these dual-functioning elements into therapeutic viral vectors will potentially provide a new generation of safer and more efficient LVs for all hematopoietic stem cell gene therapy applications.

Enhanced HbF reactivation by multiplex mutagenesis of thalassemic CD34+ cells in vitro and in vivo.

Thalassemia or sickle cell patients with hereditary persistence of fetal hemoglobin (HbF) have an ameliorated clinical phenotype and, in some cases, can achieve transfusion independence. Inactivation via genome editing of γ-globin developmental suppressors, such as BCL11A or LRF/ZBTB7A, or of their binding sites, have been shown to significantly increase expression of endogenous HbF. To broaden the therapeutic window beyond a single-editing approach, we have explored combinations of cis- and trans-editing targets to enhance HbF reactivation. Multiplex mutagenesis in adult CD34+ cells was well tolerated and did not lead to any detectable defect in the cells' proliferation and differentiation, either in vitro or in vivo. The combination of 1 trans and 1 cis mutation resulted in high editing retention in vivo, coupled with almost pancellular HbF expression in NBSGW mice. The greater in vivo performance of this combination was also recapitulated using a novel helper-dependent adenoviral-CRISPR vector (HD-Ad-dualCRISPR) in CD34+ cells from ß-thalassemia patients transplanted to NBSGW mice. A pronounced increase in HbF expression was observed in human red blood cells in mice with established predominant ß0/ß0-thalassemic hemopoiesis after in vivo injection of the HD-Ad-dualCRISPR vector. Collectively, our data suggest that the combination of cis and trans fetal globin reactivation mutations has the potential to significantly increase HbF both totally and on a per cell basis over single editing and could thus provide significant clinical benefit to patients with severe ß-globin phenotype.

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.

Adaptive Immunity and Pathogenesis of Diabetes: Insights Provided by the α4-Integrin Deficient NOD Mouse.

BACKGROUND: The spontaneously diabetic "non-obese diabetic" (NOD) mouse is a faithful model of human type-1 diabetes (T1D). METHODS: Given the pivotal role of α4 integrin (CD49d) in other autoimmune diseases, we generated NOD mice with α4-deficient hematopoiesis (NOD.α4-/-) to study the role of α4 integrin in T1D. RESULTS: NOD.α4-/- mice developed islet-specific T-cells and antibodies, albeit quantitatively less than α4+ counterparts. Nevertheless, NOD.α4-/- mice were completely and life-long protected from diabetes and insulitis. Moreover, transplantation with isogeneic α4-/- bone marrow prevented progression to T1D of pre-diabetic NOD.α4+ mice despite significant pre-existing islet cell injury. Transfer of α4+/CD3+, but not α4+/CD4+ splenocytes from diabetic to NOD.α4-/- mice induced diabetes with short latency. Despite an only modest contribution of adoptively transferred α4+/CD3+ cells to peripheral blood, pancreas-infiltrating T-cells were exclusively graft derived, i.e., α4+. Microbiota of diabetes-resistant NOD.α4-/- and pre-diabetic NOD.α4+ mice were identical. Co- housed diabetic NOD.α4+ mice showed the characteristic diabetic dysbiosis, implying causality of diabetes for dysbiosis. Incidentally, NOD.α4-/- mice were protected from autoimmune sialitis. CONCLUSION: α4 is a potential target for primary or secondary prevention of T1D.

Control of fetal globin expression in man: new opportunities to challenge past discoveries.

Decades-old findings supporting origin of F cells in adult life from adult-type progenitors and the in vitro and in vivo enhancement of fetal globin under stress conditions have been juxtaposed against recent mechanistic underpinnings. An updated molecular interrogation did not debunk prior conclusions on the origin of F cells. Although fetal globin reactivation by widely diverse approaches in vitro and in response to anemic stresses in vivo is a work in progress, accumulating evidence converges toward an integrated stress response pathway. The newly uncovered developmental regulators of globin gene switching not only have provided answers to the long-awaited quest of transregulation of switching, they are also reaching a clinical threshold. Although the effect of fetal globin silencers has been robustly validated in adult cells, the response of cells at earlier developmental stages has been unclear and inadequately studied.

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.

Myocardial Infarction Does Not Accelerate Atherosclerosis in a Mouse Model of Type 1 Diabetes.

In addition to increasing the risk of an initial myocardial infarction (MI), diabetes increases the risk of a recurrent MI. Previous work suggests that an experimental MI can accelerate atherosclerosis via monocytosis. To test whether diabetes and experimental MI synergize to accelerate atherosclerosis, we performed ligation of the left anterior descending coronary artery to induce experimental MI or sham surgery in nondiabetic and diabetic mice with preexisting atherosclerosis. All mice subjected to experimental MI had significantly reduced left ventricular function. In our model, in comparisons with nondiabetic sham mice, neither diabetes nor MI resulted in monocytosis. Neither diabetes nor MI led to increased atherosclerotic lesion size, but diabetes accelerated lesion progression, exemplified by necrotic core expansion. The necrotic core expansion was dependent on monocyte recruitment, as mice with myeloid cells deficient in the adhesion molecule integrin α4 were protected from necrotic core expansion. In summary, diabetes, but not MI, accelerates lesion progression, suggesting that the increased risk of recurrent MI in diabetes is due to a higher lesional burden and/or elevated risk factors rather than the acceleration of the underlying pathology from a previous MI.

Reappraising the role of α5 integrin and the microenvironmental support in stress erythropoiesis.

We previously studied the role of ß1 integrin and some of its different α partners relevant to erythropoiesis. Although clear and consistent answers regarding the role of α4ß1 (VLA-4) were evident, the role of its companion integrin α5ß1 (VLA-5) was clouded by inconsistent outcomes in all prior publications. Furthermore, the functional consequences of integrin deficiencies only in microenvironmental (ME) cells supporting erythroid cell expansion and maturation post stress have never been explored. In the study described here, we created several additional mouse models in the aim of addressing unanswered questions regarding functional consequences of single or combined integrin deficiencies in erythroid cells or only in ME supporting cells. Our novel and expansive data solidified the intrinsic requirement of both α4 and α5 integrins in erythroid cells for their proliferative expansion and maturation in response to stress; α5 integrin alone, deleted either early in all hematopoietic cells or only in erythroid cell, has only a redundant role in proliferative expansion and is dispensable for erythroid maturation. By contrast, α4 integrin, on its own, exerts a dominant effect on timely and optimal erythroid maturation. Deficiency of both α4 and α5 integrins in ME cells, including macrophages, does not negatively influence stress response by normal erythroid cells, in great contrast to the effect of ME cells deficient in all ß1 integrins. Collectively the present data offer deeper insight into the coordination of different ß1 integrin functional activities in erythroid cells or in ME cells for optimal erythroid stress response.

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.

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.

In vivo hematopoietic stem cell gene therapy ameliorates murine thalassemia intermedia.

Current thalassemia gene therapy protocols require the collection of hematopoietic stem/progenitor cells (HSPCs), in vitro culture, lentivirus vector transduction, and retransplantation into myeloablated patients. Because of cost and technical complexity, it is unlikely that such protocols will be applicable in developing countries, where the greatest demand for a ß-thalassemia therapy lies. We have developed a simple in vivo HSPC gene therapy approach that involves HSPC mobilization and an intravenous injection of integrating HDAd5/35++ vectors. Transduced HSPCs homed back to the bone marrow, where they persisted long-term. HDAd5/35++ vectors for in vivo gene therapy of thalassemia had a unique capsid that targeted primitive HSPCs through human CD46, a relatively safe SB100X transposase-based integration machinery, a micro-LCR-driven γ-globin gene, and an MGMT(P140K) system that allowed for increasing the therapeutic effect by short-term treatment with low-dose O6-benzylguanine plus bis-chloroethylnitrosourea. We showed in "healthy" human CD46-transgenic mice and in a mouse model of thalassemia intermedia that our in vivo approach resulted in stable γ-globin expression in the majority of circulating red blood cells. The high marking frequency was maintained in secondary recipients. In the thalassemia model, a near-complete phenotypic correction was achieved. The treatment was well tolerated. This cost-efficient and "portable" approach could permit a broader clinical application of thalassemia gene therapy.

Disruption of the BCL11A Erythroid Enhancer Reactivates Fetal Hemoglobin in Erythroid Cells of Patients with ß-Thalassemia Major.

In the present report, we carried out clinical-scale editing in adult mobilized CD34+ hematopoietic stem and progenitor cells (HSPCs) using zinc-finger nuclease-mediated disruption of BCL11a to upregulate the expression of γ-globin (fetal hemoglobin). In these cells, disruption of the erythroid-specific enhancer of the BCL11A gene increased endogenous γ-globin expression to levels that reached or exceeded those observed following knockout of the BCL11A coding region without negatively affecting survival or in vivo long-term proliferation of edited HSPCs and other lineages. In addition, BCL11A enhancer modification in mobilized CD34+ cells from patients with ß-thalassemia major resulted in a readily detectable γ-globin increase with a preferential increase in G-gamma, leading to an improved phenotype and, likely, a survival advantage for maturing erythroid cells after editing. Furthermore, we documented that both normal and ß-thalassemia HSPCs not only can be efficiently expanded ex vivo after editing but can also be successfully edited post-expansion, resulting in enhanced early in vivo engraftment compared with unexpanded cells. Overall, this work highlights a novel and effective treatment strategy for correcting the ß-thalassemia phenotype by genome editing.

HDAd5/35++ Adenovirus Vector Expressing Anti-CRISPR Peptides Decreases CRISPR/Cas9 Toxicity in Human Hematopoietic Stem Cells.

We generated helper-dependent HDAd5/35++ adenovirus vectors expressing CRISPR/Cas9 for potential hematopoietic stem cells (HSCs) gene therapy of ß-thalassemia and sickle cell disease through re-activation of fetal γ-globin expression (HDAd-globin-CRISPR). The process of CRISPR/Cas9 gene transfer using these vectors was not associated with death of human CD34+ cells and did not affect their in vitro expansion and erythroid differentiation. However, functional assays for primitive HSCs, e.g., multi-lineage progenitor colony formation and engraftment in irradiated NOD/Shi-scid/interleukin-2 receptor γ (IL-2Rγ) null (NSG) mice, revealed toxicity of HDAd-globin-CRISPR vectors related to the prolonged expression and activity of CRISPR/Cas9. To control the duration of CRISPR/Cas9 activity, we generated an HDAd5/35++ vector that expressed two anti-CRISPR (Acr) peptides (AcrII4 and AcrII2) capable of binding to the CRISPR/Cas9 complex (HDAd-Acr). CD34+ cells that were sequentially infected with HDAd-CRISPR and HDAd-Acr engrafted at a significantly higher rate. Target site disruption frequencies in engrafted human cells were similar to those in pre-transplantation CD34+ cells, indicating that genome-edited primitive HSCs survived. In vitro differentiated HSCs isolated from transplanted mice demonstrated increased γ-globin expression as a result of genome editing. Our data indicate that the HDAd-Acr vector can be used as a tool to reduce HSC cytotoxicity of the CRISPR/Cas9 complex.

Integrating HDAd5/35++ Vectors as a New Platform for HSC Gene Therapy of Hemoglobinopathies.

We generated an integrating, CD46-targeted, helper-dependent adenovirus HDAd5/35++ vector system for hematopoietic stem cell (HSC) gene therapy. The ∼12-kb transgene cassette included a ß-globin locus control region (LCR)/promoter driven human γ-globin gene and an elongation factor alpha-1 (EF1α)-mgmtP140K expression cassette, which allows for drug-controlled increase of γ-globin-expressing erythrocytes. We transduced bone marrow lineage-depleted cells from human CD46-transgenic mice and transplanted them into lethally irradiated recipients. The percentage of γ-globin-positive cells in peripheral blood erythrocytes in primary and secondary transplant recipients was stable and greater than 90%. The γ-globin level was 10%-20% of adult mouse globin. Transgene integration, mediated by a hyperactive Sleeping Beauty SB100x transposase, was random, without a preference for genes. A second set of studies was performed with peripheral blood CD34+ cells from mobilized donors. 10 weeks after transplantation of transduced cells, human cells were harvested from the bone marrow and differentiated ex vivo into erythroid cells. Erythroid cells expressed γ-globin at a level of 20% of adult α-globin. Our studies suggest that HDAd35++ vectors allow for efficient transduction of long-term repopulating HSCs and high-level, almost pancellular γ-globin expression in erythrocytes. Furthermore, our HDAd5/35++ vectors have a larger insert capacity and a safer integration pattern than currently used lentivirus vectors.