In vivo safety and antitumor efficacy of bifunctional small hairpin RNAs specific for the human Stathmin 1 oncoprotein.

Bifunctional small hairpin RNAs (bi-shRNAs) are functional miRNA/siRNA composites that are optimized for posttranscriptional gene silencing through concurrent mRNA cleavage-dependent and -independent mechanisms (Rao et al., 2010 ). We have generated a novel bi-shRNA using the miR30 scaffold that is highly effective for knockdown of human stathmin (STMN1) mRNA. STMN1 overexpression well documented in human solid cancers correlates with their poor prognosis. Transfection with the bi-shSTMN1-encoding expression plasmid (pbi-shSTMN1) markedly reduced CCL-247 human colorectal cancer and SK-Mel-28 melanoma cell growth in vitro (Rao et al., 2010 ). We now examine in vivo the antitumor efficacy of this RNA interference-based approach with human tumor xenografted athymic mice. A single intratumoral (IT) injection of pbi-shSTMN1 (8 µg) reduced CCL-247 tumor xenograft growth by 44% at 7 days when delivered as a 1,2-dioleoyl-3-trimethyl-ammoniopropane:cholesterol liposomal complex. Extended growth reductions (57% at day 15; p < 0.05) were achieved with three daily treatments of the same construct. STMN1 protein reduction was confirmed by immunoblot analysis. IT treatments with pbi-shSTMN1 similarly inhibited the growth of tumorgrafts derived from low-passage primary melanoma (≥70% reduction for 2 weeks) and abrogated osteosarcoma tumorgraft growth, with the mature bi-shRNA effector molecule detectable for up to 16 days after last injection. Antitumor efficacy was evident for up to 25 days posttreatment in the melanoma tumorgraft model. The maximum tolerated dose by IT injection of >92 µg (Human equivalent dose [HED] of >0.3 mg/kg) in CCL-247 tumor xenograft-bearing athymic mice was ∼10-fold higher than the extrapolated IC(50) of 9 µg (HED of 0.03 mg/kg). Healthy, immunocompetent rats were used as biorelevant models for systemic safety assessments. The observed maximum tolerated dose of <100 µg for intravenously injected pbi-shSTMN1 (mouse equivalent of <26.5 µg; HED of <0.09 mg/kg) confirmed systemic safety of the therapeutic dose, hence supporting early-phase assessments of clinical safety and preliminary efficacy.

Hereditary inclusion body myopathy: single patient response to intravenous dosing of GNE gene lipoplex.

Hereditary inclusion body myopathy (HIBM) is an autosomal recessive adult-onset myopathy due to mutations in the GNE (UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase) gene. Affected patients have no therapeutic options. We have previously demonstrated in preclinical testing the ability to safely correct GNE gene function through liposomal delivery of the wild-type GNE gene. Results were verified in a single patient treated by intravenous infusion of GNE gene lipoplex. A single patient (patient 001) with severe HIBM treated with a compassionate investigational new drug received seven doses of GNE gene lipoplex via intravenous infusion at the following doses: 0.4, 0.4, 1.0, 4.0, 5.0, 6.0, and 7.0 mg of DNA. GNE transgene expression, downstream induction of sialic acid, safety, and muscle function were evaluated. Transient low-grade fever, myalgia, tachycardia, transaminase elevation, hyponatremia, and hypotension were observed after infusion of each dose of GNE gene lipoplex. Quadriceps muscle expression of the delivered GNE, plasmid, and RNA was observed 24 hr after the 5.0-mg dose and at significantly greater levels 72 hr after the 7.0-mg infusion in comparison with expression in quadriceps muscle immediately before infusion. Sialic acid-related proteins were increased and stabilization in the decline of muscle strength was observed. We conclude that clinical safety and activity have been demonstrated with intravenous infusion of GNE gene lipoplex. Further assessment will involve a phase I trial of intravenous administration of GNE gene lipoplex in individuals with less advanced HIBM with more muscle function.

Hereditary inclusion body myopathy: single patient response to GNE gene Lipoplex therapy.

BACKGROUND: Hereditary inclusion body myopathy (HIBM) is an autosomal recessive adult onset myopathy. It is characterized by mutations of the GNE (UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase) gene. Afflicted patients have no therapeutic options. In preclinical testing, we have previously demonstrated the ability to correct GNE gene function and the safety of delivery of wild type GNE gene using a liposomal delivery vehicle. METHODS: A single patient (subject #001) with severe HIBM treated by compassionate investigational new drug received four doses of GNE gene Lipoplex via intramuscular injection. GNE transgene expression, downstream induction of sialic acid, safety and muscle function were evaluated. RESULTS: Significant durable improvement in locoregional skeletal muscle function was observed in the injected left extensor carpi radialis longus of #001 in correlation with GNE transgene upregulation and local induction of sialic acid. Other than transient low grade fever and pain at the injection site, no significant toxicity was observed. CONCLUSIONS: Proof of principle for manufacturing of 'clinical grade' GNE gene Lipoplex, clinical safety and activity are demonstrated with GNE gene Lipoplex. Further assessment will involve intravenous administration and subsequent phase I trial involving additional but less severely afflicted HIBM patients.

A phase I study of telomerase-specific replication competent oncolytic adenovirus (telomelysin) for various solid tumors.

A phase I clinical trial was conducted to determine the clinical safety of Telomelysin, a human telomerase reverse transcriptase (hTERT) promoter driven modified oncolytic adenovirus, in patients with advanced solid tumors. A single intratumoral injection (IT) of Telomelysin was administered to three cohorts of patients (1 x 10(10), 1 x 10(11), 1 x 10(12) viral particles). Safety, response and pharmacodynamics were evaluated. Sixteen patients with a variety of solid tumors were enrolled. IT of Telomelysin was well tolerated at all dose levels. Common grade 1 and 2 toxicities included injection site reactions (pain, induration) and systemic reactions (fever, chills). hTERT expression was demonstrated at biopsy in 9 of 12 patients. Viral DNA was transiently detected in plasma in 13 of 16 patients. Viral DNA was detectable in four patients in plasma or sputum at day 7 and 14 post-treatment despite below detectable levels at 24 h, suggesting viral replication. One patient had a partial response of the injected malignant lesion. Seven patients fulfilled Response Evaluation Criteria in Solid Tumors (RECIST) definition for stable disease at day 56 after treatment. Telomelysin was well tolerated. Evidence of antitumor activity was suggested.

Safety and in vivo expression of a GNE-transgene: a novel treatment approach for hereditary inclusion body myopathy-2.

Hereditary inclusion body myopathy-2 (HIBM2) is an adult-onset, muscular disease caused by mutations in the GNE gene. HIBM2-associated GNE mutations causing hyposialyation have been proposed to contribute to reduced muscle function in patients with HIBM2, though the exact cause of this disease is unknown. In the current studies we examined pre-clinical in vivo toxicity, and expression of the plasmid-based, CMV driven wild-type GNE plasmid vector. The plasmid vector was injected intramuscularly (IM) or systemically (IV) into BALB/c mice, following encapsulation in a cationic liposome (DOTAP:Cholesterol). Single IM injections of the GNE-lipoplex at 40 microg did not produce overt toxicity or deaths, indicating that the no observable adverse effect level (NOAEL) dose for IM injection was >or=40 microg. Single intravenous (IV) infusion of GNE-lipoplex was lethal in 33% of animals at 100 microg dose, with a small proportion of animals in the 40 microg cohort demonstrating transient toxicity. Thus the NOAEL dose by the IV route was greater than 10 microg and less than or equal to 40 microg. Real-time RT-qPCR analysis demonstrated recombinant human GNE mRNA expression in 100% of muscle tissues that received IM injection of 40 microg GNE-lipoplex, at 2 weeks. These results indicate that GNE-lipoplex gene transfer is safe and can produce durable transgene expression in treated muscles. Our findings support future exploration of the clinical efficacy of GNE-lipoplex for experimental gene therapy of HIBM2.

Antitumor activity of an oncolytic adenoviral-CD40 ligand (CD154) transgene construct in human breast cancer cells.

PURPOSE: CD40 ligand (CD40L, CD154) plays a central role in immunoregulation and also directly modulates epithelial cell growth and differentiation. We previously showed that the CD40 receptor is commonly expressed in primary breast cancer tissues. In this proof-of-principle study, we examined the breast cancer growth-regulatory activities of an oncolytic adenoviral construct carrying the CD40L transgene (AdEHCD40L). EXPERIMENTAL DESIGN: In vitro and in vivo evaluations were carried out on AdEHCD40L to validate selective viral replication and CD40L transgene activity in hypoxia inducing factor-1alpha and estrogen receptor-expressing human breast cancer cells. RESULTS: AdEHCD40L inhibited the in vitro growth of CD40+ human breast cancer lines (T-47D, MDA-MB-231, and BT-20) by up to 80% at a low multiplicity of infection of 1. Incorporation of the CD40L transgene reduced the effective dose needed to achieve 50% growth inhibition (ED50) by approximately 10-fold. In contrast, viral and transgene expression of AdEHCD40L, as well its cytotoxicity, was markedly attenuated in nonmalignant cells. Intratumoral injections with AdEHCD40L reduced preexisting MDA-MB-231 xenograft growth in severe combined immunodeficient mice by >99% and was significantly more effective (P<0.003) than parental virus AdEH (69%) or the recombinant CD40L protein (49%). This enhanced antitumor activity correlated with cell cycle blockade and increased apoptosis in AdEHCD40L-infected tumor cells. CONCLUSIONS: These novel findings, together with the previously known immune-activating features of CD40L, support the potential applicability of AdEHCD40L for experimental treatment of human breast cancer.

The role of CC chemokine receptor 6 in host defense in a model of invasive pulmonary aspergillosis.

RATIONALE: Invasive aspergillosis is a severe fungal infection afflicting immunocompromised patients, particularly patients with neutrophil defects. CCR6, a beta-chemokine receptor, mediates migration of dendritic cells (DCs) and several lymphocyte subsets to sites of epithelial inflammation, but its role in infections has not been examined extensively. OBJECTIVES: To test the hypothesis that CCR6-mediated leukocyte recruitment is necessary for effective host defense in neutropenic hosts with invasive pulmonary aspergillosis. METHODS: Neutropenic wild-type mice and mice with targeted deletion of CCR6 were infected with Aspergillus fumigatus. The host responses to the infection were compared in vivo and leukocyte responses to the fungus were examined in vitro. MEASUREMENTS AND MAIN RESULTS: In the context of infection, immature myeloid DCs were the major population of CCR6-expressing cells in the lungs. As compared with wild-type animals, CCR6-deficient mice developed a more severe infection when challenged with A. fumigatus conidia, as documented by a higher mortality rate and greater lung fungal burden. This was associated with reduced accumulation of DCs in the lungs. CCR6-deficient and wild-type DCs did not differ in their phagocytosis of conidia, cytokine response, or maturation in vitro. In adoptive transfer experiments, however, DCs from CCR6-deficient donors showed lesser accumulation in the lungs of infected mice as compared with wild-type cells, and transfer of wild-type, but not CCR6-deficient, DCs resulted in attenuated severity of infection in CCR6-deficient recipients. CONCLUSIONS: Taken together, these results implicate CCR6-mediated DC influx into the lung in the initial host defense in invasive aspergillosis.

Pathogenesis of a Texas feline immunodeficiency virus isolate: an emerging subtype of clade B.

We have recently provided evidence that Texas feline immunodeficiency virus (FIV-TX) isolates are an emerging subtype sharing a common ancestry with clade B isolates. Specific, pathogen-free cats were infected, intravenously, with 500, 2000 or 8000TCID(50) of the FIV-TX53 virus to study the acute stage of infection. Infection of cats resulted in lymphadenopathy at 10 days post-infection (p.i.). By 7 weeks p.i., gag specific antibody could be detected from sera of all infected cats. Virus could be detected by culturing PBMC and by nested capsid PCR. A reduction in the absolute numbers of lymphocytes and neutrophils was observed in infected cats although there was no trend identified between this reduction and the viral dose administered. By 11 weeks p.i., the CD4(+)/CD8(+) T cell ratios from all infected cats had dropped from approximately 2 to below 1. While decrease in the ratio was dependent on the viral dose, the T cell ratios of cats receiving the highest dose had significantly dropped below 1 by 4-7 weeks p.i. This decrease in the ratio was accompanied by a sharp and temporal decline in the absolute CD4(+) T cells and a slight increase in the absolute CD8(+) T cell numbers with a dramatic expansion of cells with CD8beta(low) chain expression.

Feline CD8+ cells induced with FIV infected, irradiated T cells produce multiple anti-FIV factors.

Feline immunodeficiency virus (FIV) infection in cats is the only non-primate, small animal model for HIV-AIDS. Replication of FIV has been shown to be optimally suppressed by soluble factors produced by inducer cell-stimulated feline CD8+ cells from FIV-infected cats. The nature of this dose-dependent suppression of FIV was examined. Antiviral factors, produced in serum-free medium, were shown to be either heat stable or heat labile. Suppressing activity was identified in a heparin-bound fraction and the non-bound fraction and in fractions separated by reverse-phase HPLC. The FIV suppression could not be correlated with IFN type I or II. Neither alpha nor beta chemokines were likely candidates because molecular size exclusion centrifugation indicated that the major factors were larger than 50 kD. Identified qualitative differences in the properties of the soluble suppressive activity generated from feline lymphocytes indicated that multiple factors are responsible for the non-cytolytic CD8+ T cell suppression of FIV replication.

Spontaneous T cell apoptosis in feline immunodeficiency virus (FIV)-infected cats is inhibited by IL2 and anti-B7.1 antibodies.

Lymph node (LN) T cells from feline immunodeficiency virus (FIV)-infected cats have an increased expression of B7 co-stimulatory molecules as well as their ligand CTLA4, resembling an activation phenotype shown to induce anergy and apoptosis in activated T cells. In addition, LN T cells from FIV-infected cats also show increased spontaneous apoptosis compared to uninfected animals. The apoptosis observed in these animals occurs primarily in T cells expressing B7 and CTLA4, suggesting a role for B7 and CTLA4 interactions in the induction of anergy/apoptosis. In order to investigate the role of B7 and CTLA4 interactions on T cell apoptosis in LN T cells from FIV-infected cats, we performed blocking experiments by measuring T cell apoptosis in LN T cell cultures treated with anti-feline B7.1, B7.2, and CTLA4 specific antibodies, as well as interleukin (IL)-2. The addition of IL2, the primary cytokine produced by B7/CD28 interactions, resulted in a significant decrease of T cell apoptosis in cultured LN cells as assessed by two-color flow cytometry and TUNEL assay. The addition of anti-B7.1 antibodies significantly inhibited T cell apoptosis in FIV-infected cats with low-level plasma viremia, while addition of anti-B7.2 and anti-CTLA4 antibodies had no affect. These results suggest a role of B7 signaling in the increased spontaneous apoptosis observed in LN T cells from FIV-infected animals.

B7+CTLA4+ T cells engage in T-T cell interactions that mediate apoptosis: a model for lentivirus-induced T cell depletion.

Apoptosis in lymph node (LN) T cells of feline immunodeficiency virus (FIV)-infected cats is associated with cells co-expressing B7.1 and B7.2 costimulatory molecules, and their ligand CTLA4. To study the possibility of B7.1/B7.2-CTLA4 mediated T-T interactions and the predicted induction of T cell apoptosis in vitro, costimulatory molecules were up-regulated on CD4+ and CD8+ T cells by mitogen stimulation. B7.1 expression on in vitro stimulated CD4+ and CD8+ cells increased within 24h; B7.2 and CTLA4 expression increased after 48-72 h. Apoptosis, as analyzed by terminal deoxynucleotidyl transferase (transferase nick end labeling, TUNEL)-based staining followed by three color flow cytometric analysis, correlated to the cells expressing B7 and/or CTLA4. Blocking experiments revealed that CD4+ and CD8+ T cell apoptosis could be significantly inhibited with anti-B7 antibodies. As FIV infection results in immune activation with a T cell phenotype similar to that of the in vitro activated T cells, the data support the hypothesis that the chronic expansion of B7+CTLA4+ LN T cells in infected cats allows for T-T cell interactions resulting in T cell depletion and eventually the development of AIDS.

The role of inducer cells in mediating in vitro suppression of feline immunodeficiency virus replication.

CD8+ T-cell-mediated suppression of feline immunodeficiency virus (FIV) replication has been described by several groups, although the mechanisms of activation and conditions for viral suppression vary with the methodologies. We have previously reported that CD8+ T-cell-mediated suppression of FIV replication required inducer cell stimulation of the effector cells. The focus of the present study was to examine the essential role of inducer cells required for the induction of this soluble anti-FIV activity. Both FIV-PPR-infected T cells and feline skin fibroblasts (FSF) infected with an alphavirus vector expressing FIV capsid or the irrelevant antigen lacZ, stimulated autologous or heterologous effector cells to produce supernatants that suppressed FIV replication. Thus, induction of this suppression of FIV replication did not strictly require autologous inducer cells and did not require the presence of FIV antigen. Anti-viral activity correlated with the presence of CD8+ T cells. Suppression was maximal when the inducer cells and the effector cells were in contact with each other, because separation of the inducer and effector cells by a 0.45-microm membrane reduced FIV suppression by approximately 50%. These findings emphasize the importance for membrane antigen interactions and cytokines in the optimal induction of effector cell synthesis of the soluble anti-FIV activity.

Feline immunodeficiency virus infection is characterized by B7+CTLA4+ T cell apoptosis.

The B7.1 and B7.2 costimulatory molecules on antigen-presenting cells provide second signals for regulating T cell immune responses via CD28 and cytotoxic T lymphocyte antigen 4 (CTLA4) on T cells. CD28 signals cell proliferation, whereas CTLA4 signals for anergy or apoptosis, terminating the immune response. Because T cell apoptosis and immunodeficiency is a characteristic of feline immunodeficiency virus (FIV)-infected cats, it is possible that negative T cell signaling via B7 and CTLA4 may be favored in these cats. Flow cytometry revealed high percentages of CD8+ and CD4+ cells expressing B7.1, B7.2, and CTLA4 in lymph nodes of FIV-positive cats and a large fraction of CTLA4+ T cells coexpressing B7.1 and B7.2. Three-color analysis with anti-B7.1, anti-B7.2, or anti-CTLA4 and TUNEL (terminal deoxynucleotidyl transferase nick-end-labeling) analysis revealed that apoptosis was a characteristic of B7.1+ B7.2+ CTLA4+ T cells. These data support the hypothesis that lymph node apoptosis and immune deterioration in FIV-infected cats results from chronic B7.1- and/or B7.2-CTLA4-mediated T-T interactions.