Sustained correction of bleeding disorder in hemophilia B mice by gene therapy

Mice generated by disrupting the clotting factor IX gene exhibit severe bleeding disorder and closely resemble the phenotype seen in hemophilia B patients. Here we demonstrate that a single intraportal injection of a recombinant adeno-associated virus (AAV) vector encoding canine factor IX cDNA under the control of a liver-specific enhancer/promoter leads to a long-term and complete correction of the bleeding disorder. High level expression of up to 15–20 μg/ml of canine factor IX was detected in the plasma of mice injected with 5.6 × 1011 particles of an AAV vector for >5 months. The activated partial thromboplastin time of the treated mice was fully corrected to higher than normal levels. Liver-specific expression of canine factor IX was confirmed by immunofluorescence staining, and secreted factor IX protein was identified in the mouse plasma by Western blotting. All treated mice survived the tail clip test without difficulty. Thus, a single intraportal injection of a recombinant adeno-associated virus vector expressing factor IX successfully cured the bleeding disorder of hemophilia B mice, proving the feasibility of using AAV-based vectors for liver-targeted gene therapy of genetic diseases.

Hepatocyte gene therapy in a large animal: A neonatal bovine model of citrullinemia

The development of gene-replacement therapy for inborn errors of metabolism has been hindered by the limited number of suitable large-animal models of these diseases and by inadequate methods of assessing the efficacy of treatment. Such methods should provide sensitive detection of expression in vivo and should be unaffected by concurrent pharmacologic and dietary regimens. We present the results of studies in a neonatal bovine model of citrullinemia, an inborn error of urea-cycle metabolism characterized by deficiency of argininosuccinate synthetase and consequent life-threatening hyperammonemia. Measurements of the flux of nitrogen from orally administered 15NH4 to [15N]urea were used to determine urea-cycle activity in vivo. In control animals, these isotopic measurements proved to be unaffected by pharmacologic treatments. Systemic administration of a first-generation E1-deleted adenoviral vector expressing human argininosuccinate synthetase resulted in transduction of hepatocytes and partial correction of the enzyme defect. The isotopic method showed significant restoration of urea synthesis. Moreover, the calves showed clinical improvement and normalization of plasma glutamine levels after treatment. The results show the clinical efficacy of treating a large-animal model of an inborn error of hepatocyte metabolism in conjunction with a method for sensitively measuring correction in vivo. These studies will be applicable to human trials of the treatment of this disorder and other related urea-cycle disorders.

Photodynamic therapy results in induction of WAF1/CIP1/P21 leading to cell cycle arrest and apoptosis

Photodynamic therapy (PDT) is a promising new modality that utilizes a combination of a photosensitizing chemical and visible light for the management of a variety of solid malignancies. The mechanism of PDT-mediated cell killing is not well defined. We investigated the involvement of cell cycle regulatory events during silicon phthalocyanine (Pc4)-PDT-mediated apoptosis in human epidermoid carcinoma cells A431. PDT resulted in apoptosis, inhibition of cell growth, and G0-G1 phase arrest of the cell cycle, in a time-dependent fashion. Western blot analysis revealed that PDT results in an induction of the cyclin kinase inhibitor WAF1/CIP1/p21, and a down-regulation of cyclin D1 and cyclin E, and their catalytic subunits cyclin-dependent kinase (cdk) 2 and cdk6. The treatment also resulted in a decrease in kinase activities associated with all the cdks and cyclins examined. PDT also resulted in (i) an increase in the binding of cyclin D1 and cdk6 toward WAF1/CIP1/p21, and (ii) a decrease in the binding of cyclin D1 toward cdk2 and cdk6. The binding of cyclin E and cdk2 toward WAF1/CIP1/p21, and of cyclin E toward cdk2 did not change by the treatment. These data suggest that PDT-mediated induction of WAF1/CIP1/p21 results in an imposition of artificial checkpoint at G1 → S transition thereby resulting in an arrest of cells in G0-G1 phase of the cell cycle through inhibition in the cdk2, cdk6, cyclin D1, and cyclin E. We suggest that this arrest is an irreversible process and the cells, unable to repair the damages, ultimately undergo apoptosis.

Gene therapy in allergic encephalomyelitis using myelin basic protein-specific T cells engineered to express latent transforming growth factor-β1

A myelin basic protein (MBP)-specific BALB/c T helper 1 (Th1) clone was transduced with cDNA for murine latent transforming growth factor-β1 (TGF-β1) by coculture with fibroblasts producing a genetically engineered retrovirus. When SJL x BALB/c F1 mice, immunized 12–15 days earlier with proteolipid protein in complete Freund’s adjuvant, were injected with 3 × 106 cells from MBP-activated untransduced cloned Th1 cells, the severity of experimental allergic encephalomyelitis (EAE) was slightly increased. In contrast, MBP-activated (but not resting) latent TGF-β1-transduced T cells significantly delayed and ameliorated EAE development. This protective effect was negated by simultaneously injected anti-TGF-β1. The transduced cells secreted 2–4 ng/ml of latent TGF-β1 into their culture medium, whereas control cells secreted barely detectable amounts. mRNA profiles for tumor necrosis factor, lymphotoxin, and interferon-γ were similar before and after transduction; interleukin-4 and -10 were absent. TGF-β1-transduced and antigen-activated BALB/c Th1 clones, specific for hemocyanin or ovalbumin, did not ameliorate EAE. Spinal cords from mice, taken 12 days after receiving TGF-β1-transduced, antigen-activated cells, contained detectable amounts of TGF-β1 cDNA. We conclude that latent TGF-β1-transduced, self-reactive T cell clones may be useful in the therapy of autoimmune diseases.

In vivo inhibition of rat stellate cell activation by soluble transforming growth factor β type II receptor: A potential new therapy for hepatic fibrosis

Transforming growth factor β (TGF-β) is a well characterized cytokine that appears to play a major role in directing the cellular response to injury, driving fibrogenesis, and, thus, potentially underlying the progression of chronic injury to fibrosis. In this study, we report the use of a novel TGF-β receptor antagonist to block fibrogenesis induced by ligation of the common bile duct in rats. The antagonist consisted of a chimeric IgG containing the extracellular portion of the TGF-β type II receptor. This “soluble receptor” was infused at the time of injury; in some experiments it was given at 4 days after injury, as a test of its ability to reverse fibrogenesis. The latter was assessed by expression of collagen, both as the mRNA in stellate cells isolated from control or injured liver and also by quantitative histochemistry of tissue sections. When the soluble receptor was administered at the time of injury, collagen I mRNA in stellate cells from the injured liver was 26% of that from animals receiving control IgG (P < 0.0002); when soluble receptor was given after injury induction, collagen I expression was 35% of that in control stellate cells (P < 0.0001). By quantitative histochemistry, hepatic fibrosis in treated animals was 55% of that in controls. We conclude that soluble TGF-β receptor is an effective inhibitor of experimental fibrogenesis in vivo and merits clinical evaluation as a novel agent for controlling hepatic fibrosis in chronic liver injury.

MLL is fused to CBP, a histone acetyltransferase, in therapy-related acute myeloid leukemia with a t(11;16)(q23;p13.3)

The recurring translocation t(11;16)(q23;p13.3) has been documented only in cases of acute leukemia or myelodysplasia secondary to therapy with drugs targeting DNA topoisomerase II. We show that the MLL gene is fused to the gene that codes for CBP (CREB-binding protein), the protein that binds specifically to the DNA-binding protein CREB (cAMP response element-binding protein) in this translocation. MLL is fused in-frame to a different exon of CBP in two patients producing chimeric proteins containing the AT-hooks, methyltransferase homology domain, and transcriptional repression domain of MLL fused to the CREB binding domain or to the bromodomain of CBP. Both fusion products retain the histone acetyltransferase domain of CBP and may lead to leukemia by promoting histone acetylation of genomic regions targeted by the MLL AT-hooks, leading to transcriptional deregulation via aberrant chromatin organization. CBP is the first partner gene of MLL containing well defined structural and functional motifs that provide unique insights into the potential mechanisms by which these translocations contribute to leukemogenesis.

A factor IX-deficient mouse model for hemophilia B gene therapy

We have generated a mouse where the clotting factor IX (FIX) gene has been disrupted by homologous recombination. The FIX nullizygous (−/−) mouse was devoid of factor IX antigen in plasma. Consistent with the bleeding disorder, the factor IX coagulant activities for wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice were 92%, 53%, and <5%, respectively, in activated partial thromboplastin time assays. Plasma factor IX activity in the deficient mice (−/−) was restored by introducing wild-type murine FIX gene via adenoviral vectors. Thus, these factor IX-deficient mice provide a useful animal model for gene therapy studies of hemophilia B.

Prolonged production of NADPH oxidase-corrected granulocytes after gene therapy of chronic granulomatous disease

Little is known about the potential for engraftment of autologous hematopoietic stem cells in human adults not subjected to myeloablative conditioning regimens. Five adult patients with the p47phox deficiency form of chronic granulomatous disease received intravenous infusions of autologous CD34+ peripheral blood stem cells (PBSCs) that had been transduced ex vivo with a recombinant retrovirus encoding normal p47phox. Although marrow conditioning was not given, functionally corrected granulocytes were detectable in peripheral blood of all five patients. Peak correction occurred 3–6 weeks after infusion and ranged from 0.004 to 0.05% of total peripheral blood granulocytes. Corrected cells were detectable for as long as 6 months after infusion in some individuals. Thus, prolonged engraftment of autologous PBSCs and continued expression of the transduced gene can occur in adults without conditioning. This trial also piloted the use of animal protein-free medium and a blood-bank-compatible closed system of gas-permeable plastic containers for culture and transduction of the PBSCs. These features enhance the safety of PBSCs directed gene therapy.

Homogeneous immunoconjugates for boron neutron-capture therapy: Design, synthesis, and preliminary characterization

The application of immunoprotein-based targeting strategies to the boron neutron-capture therapy of cancer poses an exceptional challenge, because viable boron neutron-capture therapy by this method will require the efficient delivery of 103 boron-10 atoms by each antigen-binding protein. Our recent investigations in this area have been focused on the development of efficient methods for the assembly of homogeneous immunoprotein conjugates containing the requisite boron load. In this regard, engineered immunoproteins fitted with unique, exposed cysteine residues provide attractive vehicles for site-specific modification. Additionally, homogeneous oligomeric boron-rich phosphodiesters (oligophosphates) have been identified as promising conjugation reagents. The coupling of two such boron-rich oligophosphates to sulfhydryls introduced to the CH2 domain of a chimeric IgG3 has been demonstrated. The resulting boron-rich immunoconjugates are formed efficiently, are readily purified, and have promising in vitro and in vivo characteristics. Encouragingly, these studies showed subtle differences in the properties of the conjugates derived from the two oligophosphate molecules studied, providing a basis for the application of rational design to future work. Such subtle details would not have been as readily discernible in heterogeneous conjugates, thus validating the rigorous experimental design employed here.

Synergistic inhibition of tumor growth in a murine mammary adenocarcinoma model by combinational gene therapy using IL-12, pro-IL-18, and IL-1β converting enzyme cDNA

We report here that a cancer gene therapy protocol using a combination of IL-12, pro-IL-18, and IL-1β converting enzyme (ICE) cDNA expression vectors simultaneously delivered via gene gun can significantly augment antitumor effects, evidently by generating increased levels of bioactive IL-18 and consequently IFN-γ. First, we compared the levels of IFN-γ secreted by mouse splenocytes stimulated with tumor cells transfected with various test genes, including IL-12 alone; pro-IL-18 alone; pro-IL-18 and ICE; IL-12 and pro-IL-18; and IL-12, pro-IL-18, and ICE. Among these treatments, the combination of IL-12, pro-IL-18, and ICE cDNA resulted in the highest level of IFN-γ production from splenocytes in vitro, and similar results were obtained when these same treatments were delivered to the skin of a mouse by gene gun and IFN-γ levels were measured at the skin transfection site in vivo. Furthermore, the triple gene combinatorial gene therapy protocol was the most effective among all tested groups at suppressing the growth of TS/A (murine mammary adenocarcinoma) tumors previously implanted intradermally at the skin site receiving DNA transfer by gene gun on days 6, 8, 10, and 12 after tumor implantation. Fifty percent of mice treated with the combined three-gene protocol underwent complete tumor regression. In vivo depletion experiments showed that this antitumor effect was CD8+ T cell-mediated and partially IFN-γ-dependent. These results suggest that a combinatorial gene therapy protocol using a mixture of IL-12, pro-IL-18, and ICE cDNAs can confer potent antitumor activities against established TS/A tumors via cytotoxic CD8+ T cells and IFN-γ-dependent pathways.

Concomitant combination therapy for HIV infection preferable over sequential therapy with 3TC and non-nucleoside reverse transcriptase inhibitors

Exposure to 3TC of HIV-1 mutant strains containing non-nucleoside reverse transcriptase inhibitor (NNRTI)-specific mutations in their reverse transcriptase (RT) easily selected for double-mutant viruses that had acquired the characteristic 184-Ile mutation in their RT in addition to the NNRTI-specific mutations. Conversely, exposure of 3TC-resistant 184-Val mutant HIV-1 strains to nine different NNRTIs resulted in the rapid emergence of NNRTI-resistant virus strains at a time that was not more delayed than when wild-type HIV-1(IIIB) was exposed to the same compounds. The RTs of these resistant virus strains had acquired the NNRTI-characteristic mutations in addition to the preexisting 184-Val mutation. Surprisingly, when the 184-Ile mutant HIV-1 was exposed to a variety of NNRTIs, the 188-His mutation invariably occurred concomitantly with the 184-Ile mutation in the HIV-1 RT. Breakthrough of this double-mutant virus was markedly accelerated as compared with the mutant virus selected from the wild-type or 184-Val mutant HIV-1 strain. The double (184-Ile + 188-His) mutant virus showed a much more profound resistance profile against the NNRTIs than the 188-His HIV-1 mutant. In contrast with the sequential chemotherapy, concomitant combination treatment of HIV-1-infected cells with 3TC and a variety of NNRTIs resulted in a dramatic delay of virus breakthrough and resistance development.

Presence of an inducible HIV-1 latent reservoir during highly active antiretroviral therapy

Although highly active antiretroviral therapy (HAART) in the form of triple combinations of drugs including protease inhibitors can reduce the plasma viral load of some HIV-1-infected individuals to undetectable levels, it is unclear what the effects of these regimens are on latently infected CD4+ T cells and what role these cells play in the persistence of HIV-1 infection in individuals receiving such treatment. The present study demonstrates that highly purified CD4+ T cells from 13 of 13 patients receiving HAART with an average treatment time of 10 months and with undetectable (<500 copies HIV RNA/ml) plasma viremia by a commonly used bDNA assay carried integrated proviral DNA and were capable of producing infectious virus upon cellular activation in vitro. Phenotypic analysis of HIV-1 produced by activation of latently infected CD4+ T cells revealed the presence in some patients of syncytium-inducing virus. In addition, the presence of unintegrated HIV-1 DNA in infected resting CD4+ T cells from patients receiving HAART, even those with undetectable plasma viremia, suggests persistent active virus replication in vivo.

Interferon-β gene therapy inhibits tumor formation and causes regression of established tumors in immune-deficient mice

Despite the potential of type 1 interferons (IFNs) for the treatment of cancer, clinical experience with IFN protein therapy of solid tumors has been disappointing. IFN-β has potent antiproliferative activity against most human tumor cells in vitro in addition to its known immunomodulatory activities. The antiproliferative effect, however, relies on IFN-β concentrations that cannot be achieved by parenteral protein administration because of rapid protein clearance and systemic toxicities. We demonstrate here that ex vivo IFN-β gene transduction by a replication-defective adenovirus in as few as 1% of implanted cells blocked tumor formation. Direct in vivo IFN-β gene delivery into established tumors generated high local concentrations of IFN-β, inhibited tumor growth, and in many cases caused complete tumor regression. Because the mice were immune-deficient, it is likely that the anti-tumor effect was primarily through direct inhibition of tumor cell proliferation and survival. Based on these studies, we argue that local IFN-β gene therapy with replication-defective adenoviral vectors might be an effective treatment for some solid tumors.

Stochastic processes strongly influence HIV-1 evolution during suboptimal protease-inhibitor therapy

It has long been assumed that HIV-1 evolution is best described by deterministic evolutionary models because of the large population size. Recently, however, it was suggested that the effective population size (Ne) may be rather small, thereby allowing chance to influence evolution, a situation best described by a stochastic evolutionary model. To gain experimental evidence supporting one of the evolutionary models, we investigated whether the development of resistance to the protease inhibitor ritonavir affected the evolution of the env gene. Sequential serum samples from five patients treated with ritonavir were used for analysis of the protease gene and the V3 domain of the env gene. Multiple reverse transcription–PCR products were cloned, sequenced, and used to construct phylogenetic trees and to calculate the genetic variation and Ne. Genotypic resistance to ritonavir developed in all five patients, but each patient displayed a unique combination of mutations, indicating a stochastic element in the development of ritonavir resistance. Furthermore, development of resistance induced clear bottleneck effects in the env gene. The mean intrasample genetic variation, which ranged from 1.2% to 5.7% before treatment, decreased significantly (P < 0.025) during treatment. In agreement with these findings, Ne was estimated to be very small (500–15,000) compared with the total HIV-1 RNA copy number. This study combines three independent observations, strong population bottlenecking, small Ne, and selection of different combinations of protease-resistance mutations, all of which indicate that HIV-1 evolution is best described by a stochastic evolutionary model.

Specific therapy regimes could lead to long-term immunological control of HIV

We use mathematical models to study the relationship between HIV and the immune system during the natural course of infection and in the context of different antiviral treatment regimes. The models suggest that an efficient cytotoxic T lymphocyte (CTL) memory response is required to control the virus. We define CTL memory as long-term persistence of CTL precursors in the absence of antigen. Infection and depletion of CD4+ T helper cells interfere with CTL memory generation, resulting in persistent viral replication and disease progression. We find that antiviral drug therapy during primary infection can enable the development of CTL memory. In chronically infected patients, specific treatment schedules, either including deliberate drug holidays or antigenic boosts of the immune system, can lead to a re-establishment of CTL memory. Whether such treatment regimes would lead to long-term immunologic control deserves investigation under carefully controlled conditions.