In vivo enrichment of genetically manipulated platelets corrects the murine hemophilic phenotype and induces immune tolerance even using a low multiplicity of infection.

BACKGROUND: Our previous studies have demonstrated that platelet-specific gene delivery to hematopoietic stem cells can induce sustained therapeutic levels of platelet factor VIII (FVIII) expression in mice with hemophilia A. OBJECTIVE: In this study, we aimed to enhance platelet FVIII expression while minimizing potential toxicities. METHODS: A novel lentiviral vector (LV), which harbors dual genes, the FVIII gene driven by the αIIb promoter (2bF8) and a drug-resistance gene, the MGMT(P140K) cassette, was constructed. Platelet FVIII expression in mice with hemophilia A was introduced by transduction of hematopoietic stem cells and transplantation. The recipients were treated with O(6)-benzylguanine followed by 1,3-bis-2 chloroethyl-1-nitrosourea monthly three or four times. Animals were analyzed by using polymerase chain reaction (PCR), quantitative PCR, FVIII:C assays, and inhibitor assays. Phenotypic correction was assessed by tail clipping tests and rotational thromboelastometry analysis. RESULTS: Even using a low multiplicity of infection of 1 and a non-myeloablative conditioning regimen, after in vivo selection, the levels of platelet FVIII expression in recipients increased to 4.33 ± 5.48 mU per 10(8) platelets (n = 16), which were 19.7-fold higher than the levels obtained from the recipients before treatment. Quantitative PCR results confirmed that 2bF8/MGMT-LV-transduced cells were effectively enriched after drug-selective treatment. Fifteen of 16 treated animals survived tail clipping. Blood loss and whole blood clotting time were normalized in the treated recipients. Notably, no anti-FVIII antibodies were detected in the treated animals even after recombinant human B-domain deleted FVIII challenge. CONCLUSION: we have established an effective in vivo selective system that allows us to enrich 2bF8LV-transduced cells, enhancing platelet FVIII expression while reducing the potential toxicities associated with platelet gene therapy.

C560Rß3 caused platelet integrin αII b ß3 to bind fibrinogen continuously, but resulted in a severe bleeding syndrome and increased murine mortality.

BACKGROUND AND OBJECTIVES: ß(3)-Deficient megakaryocytes were modified by human ß(3)-lentivirus transduction and transplantation to express sufficient levels of a C560Rß(3) amino acid substitution, for investigation of how an activated αII b ß(3) conformation affects platelets in vivo in mice. PATIENT/METHODS: As in our previous report of an R560ß(3) mutation in a patient with Glanzmann thrombasthenia, R560ß(3) murine platelets spontaneously bound antibody that only recognizes activated αII b ß3 bound to its ligand, fibrinogen. RESULTS: With this murine model, we showed that αII b -R560ß3 mutation-mediated continuous binding of fibrinogen occurred in the absence of P-selectin surface expression, indicating that the integrin was in an active conformation, although the platelets circulated in a quiescent manner. Remarkably, only 35% of R560ß(3) 'mutant' mice survived for 6 months after transplantation, whereas 87% of C560ß(3) 'wild-type' mice remained alive. Pathologic examination revealed that R560ß(3) mice had enlarged spleens with extramedullary hematopoiesis and increased hemosiderin, indicating hemorrhage. R560ß(3) megakaryocytes and platelets showed abnormal morphology and irregular granule distribution. Interestingly, R560ß(3) washed platelets could aggregate upon simultaneous addition of fibrinogen and physiologic agonists, but aggregation failed when platelets were exposed to fibrinogen before activation in vitro and in vivo. CONCLUSIONS: The results demonstrate that continuous occupancy of αIIb ß3 with fibrinogen disrupts platelet structure and function, leading to hemorrhagic death consistent with Glanzmann thrombasthenia rather than a thrombotic state.

Lentivirus-mediated platelet gene therapy of murine hemophilia A with pre-existing anti-factor VIII immunity.

BACKGROUND: The development of inhibitory antibodies, referred to as inhibitors, against exogenous factor VIII in a significant subset of patients with hemophilia A remains a persistent challenge to the efficacy of protein replacement therapy. Our previous studies using the transgenic approach provided proof-of-principle that platelet-specific expression could be successful in treating hemophilia A in the presence of inhibitory antibodies. OBJECTIVE: To investigate a clinically translatable approach for platelet gene therapy of hemophilia A with pre-existing inhibitors. METHODS: Platelet FVIII expression in preimmunized FVIII(null) mice was introduced by transplantation of lentivirus-transduced bone marrow or enriched hematopoietic stem cells. FVIII expression was determined with a chromogenic assay. The transgene copy number per cell was quantitated with real-time PCR. Inhibitor titer was measured with the Bethesda assay. Phenotypic correction was assessed by the tail clipping assay and an electrolytically induced venous injury model. Integration sites were analyzed with linear amplification-mediated PCR. RESULTS: Therapeutic levels of platelet FVIII expression were sustained in the long term without evoking an anti-FVIII memory response in the transduced preimmunized recipients. The tail clip survival test and the electrolytic injury model confirmed that hemostasis was improved in the treated animals. Sequential bone marrow transplants showed sustained platelet FVIII expression resulting in phenotypic correction in preimmunized secondary and tertiary recipients. CONCLUSIONS: Lentivirus-mediated platelet-specific gene transfer improves hemostasis in mice with hemophilia A with pre-existing inhibitors, indicating that this approach may be a promising strategy for gene therapy of hemophilia A even in the high-risk setting of pre-existing inhibitory antibodies.

Lentivirus-mediated platelet-derived factor VIII gene therapy in murine haemophilia A.

BACKGROUND: Previous studies from our laboratory have demonstrated that lineage-targeted synthesis of factor VIII (FVIII) under the direction of the platelet-specific integrin alphaIIb gene promoter (2bF8) can correct the murine haemophilia A phenotype even in the presence of high titer inhibitory antibodies in a transgenic mouse model. OBJECTIVE: In this study, we assessed the efficacy of using a genetic therapy approach to correct haemophilia A in FVIII-deficient (FVIII(null)) mice by transplantation of bone marrow (BM) transduced with a lentivirus (LV)-based gene transfer cassette encoding 2bF8. RESULTS: Functional FVIII activity (FVIII:C) was detected in platelet lysates from treated mice and the levels were similar to 2bF8 heterozygous transgenic mice. Mice transplanted with 2bF8 LV-transduced BM survived tail clipping and we did not detected inhibitory or non-inhibitory FVIII antibodies over the period of this study (11 months). Furthermore, BM transferred from the primary transplant recipients into FVIII(null) secondary recipients demonstrated sustained platelet-FVIII expression leading to correction of the haemophilia A phenotype showing that gene transfer occurred within long-term repopulating haematopoietic stem cells. CONCLUSIONS: These results demonstrate that ectopic expression of FVIII in platelets by lentivirus-mediated bone marrow transduction/transplantation may be a promising strategy for gene therapy of haemophilia A in humans.

Targeting platelet GPIbalpha transgene expression to human megakaryocytes and forming a complete complex with endogenous GPIbbeta and GPIX.

Bernard-Soulier Syndrome (BSS) is a severe congenital platelet disorder that results from a deficiency of the platelet membrane glycoprotein (GP) Ib/IX complex that is composed of four subunits (GPIbalpha, GPIbbeta, GPIX, and GPV). Mutations in either GPIbalpha, GPIbbeta, or GPIX can result in BSS with many of the known mutations occurring in GPIbalpha. In this study, we have developed a gene therapy strategy to express hemagglutinin (HA)-tagged GPIbalpha in megakaryocytes and potentially correct a hereditary deficiency. To direct GPIbalpha expression in megakaryocytic lineage cells, we designed a GPIbalpha cassette where human GPIbalpha cDNA was placed under control of the megakaryocytic/platelet-specific alphaIIb promoter and inserted into a lentiviral vector. Human CD34+ peripheral blood cells (PBC) and Dami cells were transduced with alphaIIb-HA-GPIbalpha-WPT virus. Flow cytometry analysis demonstrated that 50.1% of the megakaryocytes derived from CD34+ stem cells and 97.3% of Dami cells were transduced and expressed transgene GPIbalpha protein. Immunoprecipitation with Western blot analysis demonstrated that transgene protein associated with endogenous GPIbbeta and GPIX proteins. To address further the lineage-specific expression of the alphaIIb-HA-GPIbalpha construct, three cell lines, Dami, AtT-20 and HepG2, were transfected with GPIbalpha expression plasmids and analyzed by confocal microscopy. The results demonstrated that among these three cell lines, the tissue-specific alphaIIb promoter was active only in Dami cells. Thus, GPIbalpha can be efficiently and specifically expressed in the megakaryocytic compartment of hematopoietic cells and the transgene product associates with endogenous GPIbbeta and GPIX forming a complete complex. This strategy could potentially be utilized for gene therapy of BSS.

Induction of megakaryocytes to synthesize and store a releasable pool of human factor VIII.

von Willebrand factor (VWF) is a complex plasma glycoprotein that modulates platelet adhesion at the site of a vascular injury, and it also serves as a carrier protein for factor (F)VIII. As megakaryocytes are the only hematopoietic lineage to naturally synthesize and store VWF within alpha-granules, this study was performed to determine if expression of a FVIII transgene in megakaryocytes could lead to trafficking and storage of FVIII with VWF in platelet alpha-granules. Isolex selected CD34+ cells from human G-CSF mobilized peripheral blood cells (PBC) and murine bone marrow were transduced with a retrovirus encoding the B-domain deleted form of human FVIII (BDD-FVIII). Cells were then induced with cytokines to form a population of multiple lineages including megakaryocytes. Chromogenic analysis of culture supernatant from FVIII-transduced human cells demonstrated synthesis of functional FVIII. Treatment of cells with agonists of platelet activation (ADP, epinephrine, and thrombin receptor-activating peptide) resulted in the release of VWF antigen and active FVIII into the supernatant from transduced cells. Immunofluorescence analysis of cultured human and murine megakaryocytes revealed a punctate pattern of staining for FVIII that was consistent with staining for VWF. Electron microscopy of transduced megakaryocytes using immunogold-conjugated antibodies colocalized FVIII and VWF within the alpha-granules. FVIII retained its association with VWF in human platelets isolated from the peripheral blood of NOD/SCID mice at 2-6 weeks post-transplant of transduced human PBC. These results suggest feasibility for the development of a locally inducible secretory pool of FVIII in platelets of patients with hemophilia A.

Gene therapy for platelet disorders: studies with Glanzmann's thrombasthenia.

Current research aimed at correcting platelet defects are designed to further our knowledge in the use of hematopoietic stem cells for gene therapies of hemorrhagic disorders. Information gained from these studies may be directly applicable to treatment of disorders affecting platelets (e.g. Glanzmann's thrombasthenia, Bernard Soulier syndrome, gray platelet syndrome, and von Willebrand disease) as well as other disorders affecting distinct hematopoietic cell lineages. This work specifically addresses three questions: (i) can bone marrow stem cells be given sufficient genetic information to induce abnormal megakaryocytes to synthesize transgene products that help newly formed platelets to participate in normal hemostasis? (ii) can the newly synthesized receptor be maintained as a platelet-specific protein at therapeutic levels for a reasonable period of time? and (iii) will newly expressed proteins be tolerated by the immune system or become a target for B- and T-cell mediated immunity resulting in the premature destruction and clearing of the genetically altered megakaryocytes and platelets? Answers to these questions should indicate the feasibility of targeting platelets with genetic therapies that will in turn enable better management of patients with inherited bleeding disorders. The long-range benefit of this research will be an improved understanding of the regulation of protein expression during normal megakaryocytopoiesis, and the accumulation of additional scientific knowledge about normal platelet function and the way in which platelets and other cells recognize and interact with each other.

Expression of human factor VIII under control of the platelet-specific alphaIIb promoter in megakaryocytic cell line as well as storage together with VWF.

Hemophilia A, which results in defective or deficient factor VIII (FVIII) protein, is one of the genetic diseases that has been addressed through gene therapy trials. FVIII synthesis does not occur in normal megakaryocytes. In hemophilia patients who have inhibitors to FVIII activity, megakaryocytes could be a protected site of FVIII synthesis and subsequent release. Since von Willebrand factor (VWF) is a carrier protein for FVIII, we hypothesize that by directing FVIII synthesis to megakaryocytes, it would traffick together with VWF to storage in megakaryocyte alpha-granules and the platelets derived from these cells. Such synthesis would establish a protected, releasable alpha-granule pool of FVIII together with VWF. When platelets are activated in a region of local vascular damage, FVIII and VWF could potentially be released together to provide improved local hemostatic effectiveness. To direct FVIII expression to the megakaryocyte lineage, we designed a FVIII expression cassette where the human B-domain deleted FVIII cDNA was placed under the control of the megakaryocytic/platelet-specific glycoprotein IIb (alphaIIb) promoter. We demonstrated by means of a functional FVIII activity assay that the biosynthesis of FVIII occurred normally in Dami cells transfected with FVIII. FVIII production was higher when driven by the alphaIIb promoter compared to the CMV promoter, and was increased about 8-fold following PMA treatment of the transfected Dami cells. Immunofluorescence staining of the transfected cells showed that FVIII stored together with VWF in the granules. The data indicate that the megakaryocytic compartment of hematopoietic cells may represent a potential target of gene therapy for hemophilia A-especially in those patients who have developed inhibitors to plasma FVIII.

Megakaryocyte-targeted synthesis of the integrin beta(3)-subunit results in the phenotypic correction of Glanzmann thrombasthenia.

Glanzmann thrombasthenia is an inherited bleeding disorder characterized by qualitative or quantitative defects of the platelet-specific integrin, alphaIIbbeta(3). As a result, alphaIIbbeta(3) cannot be activated and cannot bind to fibrinogen, leading to a loss of platelet aggregation. Thrombasthenia is clinically characterized by mucocutaneous hemorrhage with episodes of intracranial and gastrointestinal bleeding. To develop methods for gene therapy of Glanzmann thrombasthenia, a murine leukemia virus (MuLV)-derived vector, -889Pl(A2)beta(3), was transduced into peripheral blood CD34(+) cells from 2 patients with thrombasthenia with defects in the beta(3) gene. The human alphaIIb promoter was used in this vector to drive megakaryocyte-targeted expression of the wild-type beta(3) subunit. Proviral DNA and alphaIIbbeta(3) biosynthesis were detected after in vitro differentiation of transduced thrombasthenic CD34(+) cells with megakaryocyte growth and development factor. Flow cytometric analysis of transduced patient samples indicated that 19% of megakaryocyte progeny expressed alphaIIbbeta(3) on the surface at 34% of normal receptor levels. Treatment of transduced megakaryocytes with a combination of agonists including epinephrine and the thrombin receptor-activating peptide induced the alphaIIbbeta(3) complex to form an activated conformation capable of binding fibrinogen as measured by PAC-1 antibody binding. Transduced cells retracted a fibrin clot in vitro similar to megakaryocytes derived from a normal nonthrombasthenic individual. These results demonstrate ex vivo phenotypic correction of Glanzmann thrombasthenia and support the potential use of hematopoietic CD34(+) cells as targets for alphaIIb promoter-driven MuLV vectors for gene therapy of platelet disorders. (Blood. 2000;95:3645-3651)

Integrin alphaIIb promoter-targeted expression of gene products in megakaryocytes derived from retrovirus-transduced human hematopoietic cells.

Megakaryocyte-specific expression of the platelet-adhesion receptor, integrin alphaIIbbeta3, is caused by the presence of regulatory elements of the alphaIIb promoter that direct high-level, selective gene transcription early in megakaryocytopoiesis. To develop methods for targeted expression of transgenes, we transduced human CD34+ peripheral blood cells with a murine leukemia virus (MuLV) vector controlled by the human integrin alphaIIb promoter (nucleotides -889 to +35). A naturally occurring cDNA encoding the Pl(A2) alloantigen form (Pro(33)) of the integrin beta3 subunit was subcloned into this construct (-889Pl(A2)beta3) and transduced into cells that endogenously synthesized Pl(A1)beta3 (Leu(33)) as a marker for detection of provirus-derived beta3. The ability of this vector to target expression of Pl(A2)beta3 to megakaryocytes was first examined in cell lines. Immunoblot analysis with human anti-Pl(A2) alloserum detected synthesis of Pl(A2)beta3 in transduced promegakaryocytic cells; however, Pl(A2)beta3 protein was not detected in transduced epithelial cells. Human hematopoietic CD34+ cells were transduced with -889Pl(A2)beta3 virions and induced to differentiate with megakaryocyte growth and development factor. A hybrid alphaIIbbeta3 complex was formed in progeny megakaryocytes where provirus-derived Pl(A2)beta3 was detected associated with endogenous alphaIIb subunit. Another alphaIIb promoter-driven MuLV vector (-889nlacZ) encoding Escherichia coli beta-galactosidase was used to demonstrate that transgene expression was selectively targeted to the megakaryocyte progeny of transduced CD34+ cells. These studies demonstrate the feasibility of using alphaIIb promoter-driven MuLV vectors for gene transfer of hematopoietic CD34+ cells to target transgene expression in developing megakaryocytes and platelets and indicate potential applications toward human gene therapy for platelet disorders.

Glanzmann thrombasthenia resulting from a single amino acid substitution between the second and third calcium-binding domains of GPIIb. Role of the GPIIb amino terminus in integrin subunit association.

To gain insight into region of the platelet GPIIb-IIIa complex involved in receptor biogenesis and function, we examined the biochemical properties of a defective GPIIb-IIIa complex from patient suffering from type II Glanzmann thrombasthenia. Flow cytometric as well as immunoblot analysis of patient platelets showed significantly reduced levels of GPIIb and GPIIIa compared with a normal control. Patient platelets, however, retained the ability to retract a fibrin clot. Sequence analysis of PCR-amplified platelet GPIIb mRNA revealed an Arg327-->His amino acid substitution between the second and third calcium-binding domains of the GPIIb heavy chain, a residue that is highly conserved among integrin alpha-subunits. The recombinant His327 form of GPIIb was found to be fully capable of associating with GPIIIa, therefore the role of the calcium-binding domains in intersubunit association was further examined by constructing amino-terminal segments of GPIIb that ended before the first, second, and third calcium-binding domains. All three fragments were found to associate with GPIIIa, demonstrating that the calcium-binding domains of GPIIb are not necessary for initial complex formation. Regions amino-terminal to the calcium-binding domains of GPIIb may play a heretofore unappreciated role in integrin subunit association.

A single amino acid substitution flanking the fourth calcium binding domain of alpha IIb prevents maturation of the alpha IIb beta 3 integrin complex.

To define specific structural domains involved in the biosynthesis and processing of integrin subunits, we examined the biosynthesis of normal and mutated forms of the platelet-specific integrin alpha IIb beta 3. Platelet mRNA was isolated from a Glanzmann thrombasthenic patient who failed to express significant levels of the glycoprotein (GP) IIb-IIIa complex on the platelet surface. Sequence analysis of polymerase chain reaction-amplified platelet GPIIb mRNA revealed a Gly418-->Asp amino acid substitution in GPIIb. Gly418 is a highly conserved residue that flanks the fourth calcium binding domain of GPIIb. Cotransfection of Asp418 GPIIb and GPIIIa plasmid constructs into COS-7 cells resulted in the accumulation of a pre-GPIIb-IIIa complex that failed to reach the cell surface, in effect recreating the thrombasthenic phenotype. Pulse-chase and endoglycosidase studies demonstrated that the biosynthetic blockade occurred in a pre-Golgi compartment. Removal of the negatively charged carboxyl group of Asp418 GPIIb, creating Ala418 GPIIb, rescued intracellular transport and surface expression of the integrin complex. Mutagenesis of a homologous Gly within the integrin alpha subunit alpha v also resulted in the failure to express alpha v beta 3 on the cell surface. These results suggest that the presence of a small, uncharged amino acid 6-8 residues amino-terminal to the calcium coordination complex is crucial for the proper folding and maturation of integrin complexes.

Developing neural crest cells in culture: correlation of cytochrome oxidase activity with SSEA-1 and dopamine-beta-hydroxylase immunoreactivity.

This study examines possible changes in energy demands by developing neural crest cells in vitro using cytochrome oxidase (C.O.) histochemistry and immunohistochemical labeling of adrenergic (autonomic) cells and primary sensory neurons. Cytochrome oxidase is a key enzyme for oxidative metabolism and energy production, and it is used as a sensitive metabolic marker for neurons in the brain and dorsal root ganglia. In primary neural crest cell cultures, C.O. staining intensities differ among 4 distinct cellular populations (sensory neurons, adrenergic cells, pigment cells, and non-neuronal neural crest cells). At all stages, pigment cells exhibit extremely low C.O. staining. Neurons (both primary sensory and adrenergic cells) have higher C.O. activity than other cell types such as non-neuronal neural crest cells. This indicates that neurons have higher energy demands and presumably higher levels of functional activity than other cell types at least under the present culture conditions. All neurons in neural crest cell cultures elevate their energy demands during development, implying that energy metabolism and functional activity increase with neuronal maturation. At all stages, early determined sensory neurons exhibit more intense C.O. staining than late-developing sensory neurons. The difference in C.O. activity between the two populations of sensory neurons may be caused by different levels of functional activity due to their different time course of development. Tetrodotoxin (TTX) at 0.5-1 microM concentrations causes a decrease in the level of C.O. activity in the early determined sensory neurons, which may be correlated with a decrease in the functional activity of these neurons. Triple staining combining C.O. histochemistry with indirect immunofluorescence of antibodies against the stage specific embryonic antigen-1 (SSEA-1, which labels quail sensory neurons) and dopamine-beta-hydroxylase (DBH, which labels adrenergic cells) distinguish the level of C.O. activity between sensory neurons and autonomic cells. DBH+ cells exhibit relatively low C.O. staining. However, the C.O. activity among SSEA-1+ neurons varies from high to low levels. In general, SSEA-1+ sensory neurons are much more C.O. reactive than DBH+ autonomic cells. This suggests that developing sensory neurons in culture may have higher spontaneous and/or synaptic activity than autonomic neurons.