Screening chimeric GAA variants in preclinical study results in hematopoietic stem cell gene therapy candidate vectors for Pompe disease.

Pompe disease is a rare genetic neuromuscular disorder caused by acid α-glucosidase (GAA) deficiency resulting in lysosomal glycogen accumulation and progressive myopathy. Enzyme replacement therapy, the current standard of care, penetrates poorly into the skeletal muscles and the peripheral and central nervous system (CNS), risks recombinant enzyme immunogenicity, and requires high doses and frequent infusions. Lentiviral vector-mediated hematopoietic stem and progenitor cell (HSPC) gene therapy was investigated in a Pompe mouse model using a clinically relevant promoter driving nine engineered GAA coding sequences incorporating distinct peptide tags and codon optimizations. Vectors solely including glycosylation-independent lysosomal targeting tags enhanced secretion and improved reduction of glycogen, myofiber, and CNS vacuolation in key tissues, although GAA enzyme activity and protein was consistently lower compared with native GAA. Genetically modified microglial cells in brains were detected at low levels but provided robust phenotypic correction. Furthermore, an amino acid substitution introduced in the tag reduced insulin receptor-mediated signaling with no evidence of an effect on blood glucose levels in Pompe mice. This study demonstrated the therapeutic potential of lentiviral HSPC gene therapy exploiting optimized GAA tagged coding sequences to reverse Pompe disease pathology in a preclinical mouse model, providing promising vector candidates for further investigation.

Tumor-Specific T-Cells Engineered to Overcome Tumor Immune Evasion Induce Clinical Responses in Patients With Relapsed Hodgkin Lymphoma.

Purpose Transforming growth factor-ß (TGF-ß) production in the tumor microenvironment is a potent and ubiquitous tumor immune evasion mechanism that inhibits the expansion and function of tumor-directed responses; therefore, we conducted a clinical study to discover the effects of the forced expression of a dominant-negative TGF-ß receptor type 2 (DNRII) on the safety, survival, and activity of infused tumor-directed T cells. Materials and Methods In a dose escalation study, eight patients with Epstein Barr virus-positive Hodgkin lymphoma received two to 12 doses of between 2 × 107 and 1.5 × 108 cells/m2 of DNRII-expressing T cells with specificity for the Epstein Barr virus-derived tumor antigens, latent membrane protein (LMP)-1 and LMP-2 (DNRII-LSTs). Lymphodepleting chemotherapy was not used before infusion. Results DNRII-LSTs were resistant to otherwise inhibitory concentrations of TGF-ß in vitro and retained their tumor antigen-specific activity. After infusion, the signal from transgenic T cells in peripheral blood increased up to 100-fold as measured by quantitative polymerase chain reaction for the transgene, with a corresponding increase in the frequency of functional LMP-specific T cells. Expansion was not associated with any acute or long-term toxicity. DNRII-LSTs persisted for up to ≥ 4 years. Four of the seven evaluable patients with active disease achieved clinical responses that were complete and ongoing in two patients at > 4 years, including in one patient who achieved only a partial response to unmodified tumor-directed T cells. Conclusion TGF-ß-resistant tumor-specific T cells safely expand and persist in patients with Hodgkin lymphoma without lymphodepleting chemotherapy before infusion. DNRII-LSTs can induce complete responses even in patients with resistant disease. Expression of DNRII may be useful for the many other tumors that exploit this potent immune evasion mechanism.

Toward a Rapid Production of Multivirus-Specific T Cells Targeting BKV, Adenovirus, CMV, and EBV from Umbilical Cord Blood.

Umbilical cord blood (CB) has emerged as an effective alternative donor source for hematopoietic stem cell transplantation. Despite this success, the prolonged duration of immune suppression following CB transplantation and the naiveté of CB T cells leave patients susceptible to viral infections. Adoptive transfer of ex vivo-expanded virus-specific T cells from CB is both feasible and safe. However, the manufacturing process of these cells is complicated, lengthy, and labor-intensive. We have now developed a simplified method to manufacture a single culture of polyclonal multivirus-specific cytotoxic T cells in less than 30 days. It eliminates the need for a live virus or transduction with a viral vector, thus making this approach widely available and GMP-applicable to target multiple viruses. The use of overlapping PepMixes as a source of antigen stimulation enable expansion of the repertoire of the T cell product to any virus of interest and make it available as a third party "off the shelf" treatment for viral infections following transplantation.

Cord blood natural killer cells expressing a dominant negative TGF-ß receptor: Implications for adoptive immunotherapy for glioblastoma.

Cord blood (CB) natural killer (NK) cells are promising effector cells for tumor immunotherapy but are currently limited by immune-suppressive cytokines in the tumor microenvironment, such as transforming growth factor (TGF-ß). We observed that TGF-ß inhibits expression of activating receptors such as NKG2D and DNAM1 and decreases killing activity against glioblastoma tumor cells through inhibition of perforin secretion. To overcome the detrimental effects of TGF-ß, we engrafted a dominant negative TGF-ß receptor II (DNRII) on CB-derived NK cells by retroviral transduction and evaluated their ability to kill glioblastoma cells in the presence of TGF-ß. After manufacture using Good Manufacturing Practice-compliant methodologies and transduction with DNRII, CB-derived DNRII-transduced NK cells expanded to clinically relevant numbers and retained both their killing ability and their secretion of interferon-γ upon activation. More important, these cells maintained both perforin expression and NKG2D/DNMA1 expression in the presence of TGF-ß allowing for recognition and killing of glioblastoma tumor cells. Hence, NK cells expressing a DNRII should have a functional advantage over unmodified NK cells in the presence of TGF-ß-secreting tumors and may be an important therapeutic approach for patients with cancer.

Human parainfluenza virus-3 can be targeted by rapidly ex vivo expanded T lymphocytes.

BACKGROUND AIMS: Human parainfluenza virus-3 (HPIV) is a common cause of respiratory infection in immunocompromised patients and currently has no effective therapies. Virus-specific T-cell therapy has been successful for the treatment or prevention of viral infections in immunocompromised patients but requires determination of T-cell antigens on targeted viruses. METHODS: HPIV3-specific T cells were expanded from peripheral blood of healthy donors using a rapid generation protocol targeting four HPIV3 proteins. Immunophenotyping was performed by flow cytometry. Viral specificity was determined by interferon (IFN)-γ ELISpot, intracellular cytokine staining and cytokine measurements from culture supernatants by Luminex assay. Cytotoxic activity was tested by 51Cr release and CD107a mobilization assays. Virus-specific T cells targeting six viruses were then produced by rapid protocol, and the phenotype of HPIV3-specific T cells was determined by immunomagnetic sorting for IFN-γ-producing cells. RESULTS: HPIV3-specific T cells were expanded from 13 healthy donors. HPIV3-specific T cells showed a CD4+ predominance (mean CD4:CD8 ratio 2.89) and demonstrated specificity for multiple HPIV3 antigens. The expanded T cells were polyfunctional based on cytokine production but only had a minor cytotoxic component. T cells targeting six viruses in a single product similarly showed HPIV3 specificity, with a predominant effector memory phenotype (CD3+/CD45RA-/CCR7-) in responder cells. DISCUSSION: HPIV3-specific T cells can be produced using a rapid ex vivo protocol from healthy donors and are predominantly CD4+ T cells with Th1 activity. HPIV3 epitopes can also be successfully targeted alongside multiple other viral epitopes in production of six-virus T cells, without loss of HPIV3 specificity. These products may be clinically beneficial to combat HPIV3 infections by adoptive T-cell therapy in immune-compromised patients.

Regulated apoptosis of genetically modified hematopoietic stem and progenitor cells via an inducible caspase-9 suicide gene in rhesus macaques.

The high risk of insertional oncogenesis reported in clinical trials using integrating retroviral vectors to genetically modify hematopoietic stem and progenitor cells (HSPCs) requires the development of safety strategies to minimize risks associated with novel cell and gene therapies. The ability to ablate genetically modified cells in vivo is desirable, should an abnormal clone emerge. Inclusion of "suicide genes" in vectors to facilitate targeted ablation of vector-containing abnormal clones in vivo is one potential safety approach. We tested whether the inclusion of the "inducible Caspase-9" (iCasp9) suicide gene in a gamma-retroviral vector facilitated efficient elimination of vector-containing HSPCs and their hematopoietic progeny in vivo long-term, in an autologous non-human primate transplantation model. Following stable engraftment of iCasp9 expressing hematopoietic cells in rhesus macaques, administration of AP1903, a chemical inducer of dimerization able to activate iCasp9, specifically eliminated vector-containing cells in all hematopoietic lineages long-term, suggesting activity at the HSPC level. Between 75% and 94% of vector-containing cells were eliminated by well-tolerated AP1903 dosing, but lack of complete ablation was linked to lower iCasp9 expression in residual cells. Further investigation of resistance mechanisms demonstrated upregulation of Bcl-2 in hematopoietic cell lines transduced with the vector and resistant to AP1903 ablation. These results demonstrate both the potential and the limitations of safety approaches using iCasp9 to HSPC-targeted gene therapy settings, in a model with great relevance to clinical development.

Thymidine kinase suicide gene-mediated ganciclovir ablation of autologous gene-modified rhesus hematopoiesis.

Despite the genotoxic complications encountered in clinical gene therapy trials for primary immunodeficiency diseases targeting hematopoietic cells with integrating vectors; this strategy holds promise for the cure of several monogenic blood, metabolic and neurodegenerative diseases. In this study, we asked whether the inclusion of a suicide gene in a standard retrovirus vector would allow elimination of vector-containing stem and progenitor cells and their progeny in vivo following transplantation, using our rhesus macaque transplantation model. Following stable engraftment with autologous CD34(+) cells transduced with a retrovirus vector encoding a highly sensitive modified Herpes simplex virus thymidine kinase SR39, the administration of the antiviral prodrug ganciclovir (GCV) was effective in completely eliminating vector-containing cells in all hematopoietic lineages in vivo. The sustained absence of vector-containing cells over time, without additional GCV administration, suggests that the ablation of TkSR39 GCV-sensitive cells occurred in the most primitive hematopoietic long-term repopulating stem or progenitor cell compartment. These results are a proof-of-concept that the inclusion of a suicide gene in integrating vectors, in addition to a therapeutic gene, can provide a mechanism for later elimination of vector-containing cells, thereby increasing the safety of gene transfer.

Contributions of gene marking to cell and gene therapies.

The first human genetic modification studies used replication-incompetent integrating vector vectors to introduce marker genes into T lymphocytes and subsequently into hematopoietic stem cells. Such studies have provided numerous insights into the biology of hematopoiesis and immune reconstitution and contributed to clinical development of gene and cell therapies. Tracking of hematopoietic reconstitution and analysis of the origin of residual malignant disease after hematopoietic transplantation has been possible via gene marking. Introduction of selectable marker genes has enabled preselection of specific T-cell populations for tumor and viral immunotherapy and reduced the threat of graft-versus-host disease, improving the survival of patients after allogeneic marrow transplantation. Marking studies in humans, murine xenografts, and large animals have helped optimize conditions for gene transfer into CD34(+) hematopoietic progenitors, contributing to the achievement of gene transfer efficiencies sufficient for clinical benefit in several serious genetic diseases such as X-linked severe combined immunodeficiency and adrenoleukodystrophy. When adverse events linked to insertional mutagenesis arose in clinical gene therapy trials for inherited immunodeficiencies, additional animal studies using gene-marking vectors have greatly increased our understanding of genotoxicity. The knowledge gained from these studies is being translated into new vector designs and clinical protocols, which we hope will continue to improve the efficiency, effectiveness and safety of these promising therapeutic approaches.

SDF-1/CXCL12 enhances in vitro replating capacity of murine and human multipotential and macrophage progenitor cells.

Hematopoietic progenitor cells (HPCs) manifest a limited self-renewal capacity, as determined by a surrogate assay involving replating capacity of single colonies in vitro with generation of secondary colonies. Stromal cell-derived factor-1 (SDF-1/CXCL12), has been implicated in regulation of hematopoiesis through its modulation of hematopoietic stem cell (HSC) and HPC migration, homing, mobilization, and survival. We used bone marrow cells from SDF-1/CXCL12 transgenic and littermate control mice, and culture of normal mouse bone marrow and human cord blood cells plated in the presence or absence of recombinant SDF-1/CXCL12 to evaluate a role for SDF-1/CXCL12 in the replating capability in vitro of multipotential [colony-forming units (CFU)-GEMM] and macrophage (CFU-M) progenitor cells. Competitive repopulating capacity of mouse HSCs was assessed in lethally irradiated mice. Transgenic or exogenous SDF-1/CXCL12 significantly enhanced numbers of secondary colonies formed from primary CFU-GEMM or CFU-M colonies. In the limited setting of our in vivo studies, the SDF-1/CXCL12 transgene did not influence HSC competitive repopulation. However, the results suggest that SDF-1/CXCL12 enhances in vitro replating/self-renewal of HPCs, which may contribute to myelopoiesis in vivo. This information may be of value to ex vivo expansion of HPCs/HSCs.

Granulocyte colony-stimulating factor prior to nonmyeloablative irradiation decreases murine host hematopoietic stem cell function and increases engraftment of donor marrow cells.

The use of nonmyeloablative conditioning prior to bone marrow transplantation is an important component of transplantation-based therapies for nonmalignant blood diseases. In this study, treatment of recipient mice with granulocyte colony-stimulating factor (G-CSF) prior to low-dose total body irradiation (LD-TBI) enhanced long-term engraftment of freshly isolated congenic marrow 1.5- to 2-fold more than treatment with LD-TBI alone. This combined regimen was also evaluated in a mouse model of X-linked chronic granulomatous disease (X-CGD), where neutrophils have a defective NADPH oxidase due to genetic deletion of the gp91(phox) subunit. Long-term engraftment of male X-CGD bone marrow cells cultured ex vivo for retroviral transduction of gp91(phox) was enhanced by approximately 40% when female X-CGD recipients were pretreated with G-CSF prior to 300 cGy. These data confirm that sequential treatment with G-CSF and LD-TBI prior to transplantation increases long-term engraftment of donor marrow, and they extend this approach to transplantation of murine donor marrow cultured ex vivo for gene transfer. Additional studies showed that the administration of G-CSF prior to LD-TBI did not alter early homing of donor marrow cells. However, the combined regimen significantly decreased the content of long-term repopulating cells in recipient marrow compared with LD-TBI alone, as assessed in competitive assays, which may contribute to the enhanced engraftment of donor marrow cells. Disclosure of potential conflicts of interest is found at the end of this article.

Rac2 concentrations in umbilical cord neutrophils.

BACKGROUND: Human newborn infants display a variety of immunodeficiencies of immaturity, including diminished neutrophil adhesion, chemotaxis, and migration. Rac2, a guanosine triphosphate-binding protein, is an essential regulator of human neutrophil migration and chemotaxis. Since human subjects and mice deficient in Rac2 display deficiencies in neutrophil functions similar to newborn infants, we postulated that newborn neutrophils may be deficient in Rac2. OBJECTIVES: The aim of the study was to measure Rac1 and Rac2 concentrations in neutrophils from umbilical cord blood. METHODS: Neutrophils from cord and adult blood were isolated, total cell lysates extracted, and Rac protein concentrations determined using Western blot analysis. RESULTS: Rac2 concentrations were significantly lower in the neutrophil protein lysates isolated from cord blood compared to adult blood despite similar levels of Rac1. CONCLUSIONS: Diminished Rac2 expression in cord blood neutrophils may contribute to the defects observed in cord blood neutrophil function.

Molecular characterization of a novel splice site mutation within the CYBB gene leading to X-linked chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a primary immunodeficiency that affects the oxidative mechanism of microbial killing of phagocytic cells. The defect is characterized by a lack or severely reduced superoxide anion (O2-) production by phagocytes. Seventy percent of CGD cases are X-linked (X-CGD) and they are caused by mutations in the gene encoding for gp91(phox), one of the two subunits of the flavocytochrome b558 of the NADPH oxidase. We identified an abnormal transcript arising from a novel splice site mutation within the gene encoding gp91(phox), which suggested that the mutation affected normal mRNA splicing. Thus, the effect of this mutation leads to the complete absence of the flavocytochrome b558 in neutrophil membranes, which caused the biochemical phenotype X91 degrees-CGD in this family. These molecular findings help to explain the early onset and severe phenotype in this X-CGD kindred.

X-Linked Chronic Granulomatous Disease: First Report of Mutations in Patients of Argentina.

BACKGROUND: Chronic granulomatous disease (CGD) is a primary immunodeficiency due to absent or decreased NADPH oxidase activity in phagocytic cells. The X-linked form of the disease (X-CGD) arises from mutations in the CYBB gene, which encodes the 91-kD glycoprotein gp91, the largest component of the oxidase. METHODS: The authors recently started the molecular characterization of X-CGD in 18 patients reported to the Argentinean Registry of Primary Immunodeficiency Diseases. The authors reviewed data from clinical records to examine the relationship of clinical presentation and the type of mutations responsible for the genotype. RESULTS: The frequency and type of infections present in these patients were similar to prior reports. However, pulmonary tuberculosis was observed in the group as well as unusual complications such as eosinophilic cystitis, hepatic abscess with cholangitis, and chronic orchitis. Eleven different mutations in the CYBB gene were identified, and seven of them were novel. The types of mutations were intronic, single-nucleotide substitution resulting in nonsense or missense codons and one or two nucleotide deletions resulting in frameshifts. Molecular studies of 18 mothers revealed X-CGD carrier status in all but 2. CONCLUSIONS: No correlation existed between the type of mutation and the clinical phenotype of the disease: the molecular defects identified resulted in no expression of the flavocytochrome b558 in patients' neutrophils, leading to the X91°-CGD phenotype. The lack of gp91 protein could explain the early onset and the severity of the clinical manifestations of CGD in this group of patients from Argentina.

Gene therapy for chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a congenital immune deficiency that is a promising therapeutic target for gene replacement into haematopoietic stem cells (HSCs). CGD results from mutations in any one of four genes encoding subunits of the superoxide-generating NADPH oxidase of phagocytes. Life-threatening, recurrent bacterial and fungal infections, as well as inflammatory granulomas, are the hallmarks of the disease. NADPH oxidase activity can be reconstituted by retroviral- or lentiviral-mediated gene transfer to human CGD marrow in vitro and in xenograft transplant models. Gene transfer studies in knockout mouse models that resemble the human disease suggest that correction of oxidase activity in a minority of phagocytes will be of clinical benefit. Phase I clinical studies in unconditioned CGD patients showed transient expression of small numbers of gene-corrected neutrophils. Areas of research at present include efforts to enhance gene transfer rates into repopulating HSCs using vectors that transduce quiescent cells, and to increase the engraftment of genetically corrected HSCs using non-myeloablative conditioning and drug resistance genes for selection.

X-linked chronic granulomatous disease: first report of mutations in patients of Argentina.

BACKGROUND: Chronic granulomatous disease (CGD) is a primary immunodeficiency due to absent or decreased NADPH oxidase activity in phagocytic cells. The X-linked form of the disease (X-CGD) arises from mutations in the CYBB gene, which encodes the 91-kD glycoprotein gp91(phox), the largest component of the oxidase. METHODS: The authors recently started the molecular characterization of X-CGD in 18 patients reported to the Argentinean Registry of Primary Immunodeficiency Diseases. The authors reviewed data from clinical records to examine the relationship of clinical presentation and the type of mutations responsible for the genotype. RESULTS: The frequency and type of infections present in these patients were similar to prior reports. However, pulmonary tuberculosis was observed in the group as well as unusual complications such as eosinophilic cystitis, hepatic abscess with cholangitis, and chronic orchitis. Eleven different mutations in the CYBB gene were identified, and seven of them were novel. The types of mutations were intronic, single-nucleotide substitution resulting in nonsense or missense codons and one or two nucleotide deletions resulting in frameshifts. Molecular studies of 18 mothers revealed X-CGD carrier status in all but 2. CONCLUSIONS: No correlation existed between the type of mutation and the clinical phenotype of the disease: the molecular defects identified resulted in no expression of the flavocytochrome b558 in patients' neutrophils, leading to the X91(o)-CGD phenotype. The lack of gp91(phox) protein could explain the early onset and the severity of the clinical manifestations of CGD in this group of patients from Argentina.

Recurrent eosinophilic cystitis in a child with chronic granulomatous disease.

Eosinophilic cystitis is an uncommon disease in children, and its association with chronic granulomatous disease (CGD) has been previously reported in only five patients. In all those patients the disease showed either a self-limited benign course or a rapid response to corticosteroid treatment. The authors describe a child with X-linked CGD who developed eosinophilic cystitis with a recurrent course and difficult therapeutic management. The authors also discuss the pathogenesis of granuloma formation in CGD and review the literature for current therapies for these complications.