Transduction of rat and human adipose-tissue derived mesenchymal stromal cells by adeno-associated viral vector serotype DJ.

Ex vivo, gene therapy is a powerful approach holding great promises for the treatment of both genetic and acquired diseases. Adeno-associated virus (AAV) vectors are a safe and efficient delivery system for modification of mesenchymal stem cells (MSC) that could maximize their therapeutic benefits. Assessment of MSC viability and functional activity after infection with new AAV serotypes is necessary, due to AAV tropism to specific cell types. We infected human and rat adipose-tissue MSC with hybrid AAV-DJ serotype vectors carrying GFP and SCF genes. GFP expression from AAV-DJ was about 1.5-fold superior to that observed with AAV-2 and lasted for at least 21 days as was evaluated by flow cytometry and fluorescence microscopy. AAV-DJ proves to be suitable for the infection of rat and human MSC with a similar efficiency. Infected MSC were still viable but showed a 25-30% growth-rate slowdown. Moreover, we found an increase of SERPINB2 mRNA expression in human MSC while expression of other oxidative stress markers and extracellular matrix proteins was not affected. These results suggest that there is a differential cellular response in MSC infected with AAV viral vectors, which should be taken into account as it can affect the expected outcome for the therapeutic application.

Neurotoxicity of HIV-1 Tat is attributed to its penetrating property.

We have recently engineered an exosomal Tat (Exo-Tat) which can activate latent HIV-1 in resting CD4+ T lymphocytes from antiretroviral treated HIV-1 infected patients. HIV-1 Tat protein can penetrate cell membrane freely and secrete into extracellular medium. Exo-Tat loses this penetrating property. HIV-1 Tat protein can damage the synaptic membranes contributing to the development of dementia in HIV-1 infected patients. To investigate whether the penetrating property attributes to synaptic damage in vivo, we have generated adeno-associated viruses AAV-Tat and AAV-Exo-Tat viruses. Vehicle control or AAV viruses (1 × 1012 GC/mouse in 200 µl PBS) were injected into Balb/cj mice via tail veins. The mRNA and protein expression levels in blood, brain, heart, intestine, kidney, liver, lung, muscle and spleen were determined on day 21. Intravenously injected AAV-Tat or AAV-Exo-Tat mainly infects liver and heart. Short-term expression of Tat or Exo-Tat doesn't change the expression levels of neuronal cytoskeletal marker ß3-tubulin and synaptic marker postsynaptic density 95 protein (PSD-95). Wild-type Tat, but not Exo-Tat, reduces the expression level of synaptic marker synaptophysin significantly in mice, indicating that penetrating property of HIV-1 Tat protein attributes to synaptic damage.

Selective Neuronal Uptake and Distribution of AAVrh8, AAV9, and AAVrh10 in Sheep After Intra-Striatal Administration.

BACKGROUND: Transgenic sheep are currently the only large animal model of Huntington's disease expressing full-length mutant human huntingtin. These transgenic sheep provide an opportunity to test adeno associated virus (AAV) therapies directly targeting the huntingtin gene. A recent study demonstrated that self-complementary (sc) AAV with artificial miRNA against human huntingtin reduced mutant human huntingtin in caudate and putamen after a single injection near the internal capsule. OBJECTIVE: To identify an AAV serotype among AAVrh8, AAV9 and AAVrh10 with the highest neuronal uptake and distribution, with no obvious cell loss in the neostriatum of the sheep. METHODS: We tested AAVrh8, AAV9 and AAVrh10 by stereotactic direct unilateral injection into the neostriatum of sheep, near the internal capsule. Four weeks after administration, we examined the viral spread and neuronal uptake of each serotype of AAV containing GFP. We compared single stranded (ss) and scAAVs. Further, we measured the distribution of AAVrh8 and AAV9 to a variety of tissues outside the brain. RESULTS: Sc AAV9 had the best combination of neuronal uptake and distribution throughout the neostriatum. scAAVrh10 demonstrated good spread, but was not taken up by neurons. scAAVrh8 demonstrated good spread, but had less neuronal uptake than AAV9. Six hours after convection-enhanced administration to the neostriatum, both AAVrh8 and AAV9 viral genomes were detected in blood, saliva, urine, feces and wool. By four weeks, viral genomes were detected in wool only. Administration of AAVrh8, AAV9 and AAVrh10 was not associated with loss of neostriatal, medium spiny neuron number as measured by DARPP32 immunohistochemistry. CONCLUSIONS: Altogether, we found scAAV9 had the best neuronal uptake and spread, showed no loss of neurons at one-month post-injection, and was not measurable in body fluids one month after injection. This information will guide future clinical experiments requiring brain injection of AAV for therapeutics for gene or miRNA deliveries in sheep transgenic for the human huntingtin gene.

Molecular Analyses Support the Safety and Activity of Retroviral Replicating Vector Toca 511 in Patients.

Purpose: Toca 511 is a gammaretroviral replicating vector encoding cytosine deaminase that selectively infects tumor cells and converts the antifungal drug 5-fluorocytosine into the antineoplastic drug 5-fluorouracil, which directly kills tumor cells and stimulates antitumor immune responses. As part of clinical monitoring of phase I clinical trials in recurrent high-grade glioma, we have performed extensive molecular analyses of patient specimens to track vector fate.Patients and Methods: Toca 511 and Toca FC (extended-release 5-fluorocytosine) have been administered to 127 high-grade glioma patients across three phase I studies. We measured Toca 511 RNA and DNA levels in available body fluids and tumor samples from patients to assess tumor specificity. We mapped Toca 511 integration sites and sequenced integrated Toca 511 genomes from patient samples with detectable virus. We measured Toca 511 levels in a diverse set of tissue samples from one patient.Results: Integrated Toca 511 is commonly detected in tumor samples and is only transiently detected in blood in a small fraction of patients. There was no believable evidence for clonal expansion of cells with integrated Toca 511 DNA, or preferential retrieval of integration sites near oncogenes. Toca 511 sequence profiles suggest most mutations are caused by APOBEC cytidine deaminases acting during reverse transcription. Tissue samples from a single whole-body autopsy affirm Toca 511 tumor selectivity.Conclusions: Toca 511 and Toca FC treatment was not associated with inappropriate integration sites and clonal expansion. The vector is tumor-selective and persistent in patients who received Toca 511 injections. Clin Cancer Res; 24(19); 4680-93. ©2018 AACR.

Genetics and Gene Therapy in Hunter Disease.

Mucopolysaccharidosis type II or Hunter syndrome is an X-linked lysosomal storage disease caused by a mutation in the gene encoding the lysosomal enzyme iduronate-2-sulfatase. The consequent enzyme deficiency causes a progressive, multisystem accumulation of glycosaminoglycans, which is the cause of the clinical manifestations involving also Central Nervous System for patients with the severe form of disease. The limits of the currently available therapies for Hunter syndrome, hematopoietic stem cell transplantation and recombinant enzyme replacement therapy, mainly regarding brain achievement, have encouraged several studies which recognized gene therapy as a potential therapeutic option for this condition. In vitro studies firstly aimed at the demonstration that viral vector- mediated IDS gene expression could lead to high levels of enzyme activity in transduced cells. The encouraging results obtained allowed the realization of many preclinical studies investigating the utilization of gene therapy vectors in animal models of Mucopolysaccharidosis II, together with a phase I clinical trial approved for Hunter patients affected by the mild form of the disease. Together to in vivo studies in which recombinant vectors are directly administered, systematically or by direct injection into Central Nervous System, also ex vivo gene therapy, consisting in transplantation of autologous hematopoietic stem cells, modified in vitro, into the animal or patient, has been tested. A wider clinical application of the results obtained so far is essential to ensure that gene therapy can be definitively validated as a therapeutic option available and usable for this rare but life-threatening disorder.

Hematopoietic Stem-Cell Gene Therapy for Cerebral Adrenoleukodystrophy.

BACKGROUND: In X-linked adrenoleukodystrophy, mutations in ABCD1 lead to loss of function of the ALD protein. Cerebral adrenoleukodystrophy is characterized by demyelination and neurodegeneration. Disease progression, which leads to loss of neurologic function and death, can be halted only with allogeneic hematopoietic stem-cell transplantation. METHODS: We enrolled boys with cerebral adrenoleukodystrophy in a single-group, open-label, phase 2-3 safety and efficacy study. Patients were required to have early-stage disease and gadolinium enhancement on magnetic resonance imaging (MRI) at screening. The investigational therapy involved infusion of autologous CD34+ cells transduced with the elivaldogene tavalentivec (Lenti-D) lentiviral vector. In this interim analysis, patients were assessed for the occurrence of graft-versus-host disease, death, and major functional disabilities, as well as changes in neurologic function and in the extent of lesions on MRI. The primary end point was being alive and having no major functional disability at 24 months after infusion. RESULTS: A total of 17 boys received Lenti-D gene therapy. At the time of the interim analysis, the median follow-up was 29.4 months (range, 21.6 to 42.0). All the patients had gene-marked cells after engraftment, with no evidence of preferential integration near known oncogenes or clonal outgrowth. Measurable ALD protein was observed in all the patients. No treatment-related death or graft-versus-host disease had been reported; 15 of the 17 patients (88%) were alive and free of major functional disability, with minimal clinical symptoms. One patient, who had had rapid neurologic deterioration, had died from disease progression. Another patient, who had had evidence of disease progression on MRI, had withdrawn from the study to undergo allogeneic stem-cell transplantation and later died from transplantation-related complications. CONCLUSIONS: Early results of this study suggest that Lenti-D gene therapy may be a safe and effective alternative to allogeneic stem-cell transplantation in boys with early-stage cerebral adrenoleukodystrophy. Additional follow-up is needed to fully assess the duration of response and long-term safety. (Funded by Bluebird Bio and others; STARBEAM ClinicalTrials.gov number, NCT01896102 ; ClinicalTrialsRegister.eu number, 2011-001953-10 .).

The relevance of coagulation factor X protection of adenoviruses in human sera.

Intravenous delivery of adenoviruses is the optimal route for many gene therapy applications. Once in the blood, coagulation factor X (FX) binds to the adenovirus capsid and protects the virion from natural antibody and classical complement-mediated neutralisation in mice. However, to date, no studies have examined the relevance of this FX/viral immune protective mechanism in human samples. In this study, we assessed the effects of blocking FX on adenovirus type 5 (Ad5) activity in the presence of human serum. FX prevented human IgM binding directly to the virus. In individual human sera samples (n=25), approximately half of those screened inhibited adenovirus transduction only when the Ad5-FX interaction was blocked, demonstrating that FX protected the virus from neutralising components in a large proportion of human sera. In contrast, the remainder of sera tested had no inhibitory effects on Ad5 transduction and FX armament was not required for effective gene transfer. In human sera in which FX had a protective role, Ad5 induced lower levels of complement activation in the presence of FX. We therefore demonstrate for the first time the importance of Ad-FX protection in human samples and highlight subject variability and species-specific differences as key considerations for adenoviral gene therapy.

Improved detection of transgene and nonviral vectors in blood.

Vector biodistribution and clearance studies are essential in the development of gene transfer medicine. To provide reliable and accurate data, protocols for vector analysis must be optimized and validated. We addressed several parameters affecting the detection of gene therapy vectors in blood. Using an in vitro system based on plasmid DNA incorporating, as a transgene, complementary DNA for human erythropoietin gene, we developed and validated a suite of real-time PCR assays for the transgene splicing sites. The most sensitive assays detected the transgene present at 0.011% of the copy number of the endogenous erythropoietin gene in human genomic DNA at 100% specificity. Plasmid linearization incorporated with PCR resulted in an increase in assay sensitivity up to 4.5-fold without compromising analysis workflow. This allowed detection of five copies of transgene in a background of 0.4 µg of genomic DNA (or 0.0035% detectable transgene copies relevant to copies of the endogenous gene). Finally, desktop assessment of 18 DNA extraction protocols was undertaken and 5 kits were evaluated experimentally for extraction of nonviral vectors from blood. Three kits reliably detected 80 copies of the transgene in a milliliter of blood. Adoption of the described protocols will enable more reliable vector analysis in gene therapy and will assist in accurate interlaboratory comparison. The methodology will also facilitate detection of gene doping in sport, a potential new form of misuse of gene transfer technology.

Prevalence and pharmacological modulation of humoral immunity to AAV vectors in gene transfer to synovial tissue.

Antibodies against adeno-associated viral (AAV) vectors are highly prevalent in humans. Both preclinical and clinical studies showed that antibodies against AAV block transduction even at low titers, particularly when the vector is introduced into the bloodstream. Here we measured the neutralizing antibody (NAb) titer against AAV serotypes 2, 5, 6 and 8 in the serum and matched synovial fluid (SF) from rheumatoid arthritis patients. The titer in the SF was lower than that in the matched plasma samples, indicating a difference in distribution of NAb to AAV depending on the body fluid compartment. This difference was more evident for AAV2, against which higher titers were measured. Of all serotypes, anti-AAV5 antibodies were the least prevalent in both the serum and SF. We next evaluated the impact of B-cell depletion on anti-AAV antibodies in rheumatoid arthritis patients who received one or two courses of the anti-CD20 antibody rituximab as part of their disease management. A drop of NAb titer was observed in a subset of those subjects carrying NAb titers ≤1:1000; however, only in a minority of subjects titers dropped below 1:5. This work provides insights into strategies to overcome the limitation of pre-existing humoral immunity to AAV vectors.

A potential role of distinctively delayed blood clearance of recombinant adeno-associated virus serotype 9 in robust cardiac transduction.

Recombinant adeno-associated virus serotype 9 (rAAV9) vectors show robust in vivo transduction by a systemic approach. It has been proposed that rAAV9 has enhanced ability to cross the vascular endothelial barriers. However, the scientific basis of systemic administration of rAAV9 and its transduction mechanisms have not been fully established. Here, we show indirect evidence suggesting that capillary walls still remain as a significant barrier to rAAV9 in cardiac transduction but not so in hepatic transduction in mice, and the distinctively delayed blood clearance of rAAV9 plays an important role in overcoming this barrier, contributing to robust cardiac transduction. We find that transvascular transport of rAAV9 in the heart is a capacity-limited slow process and occurs in the absence of caveolin-1, the major component of caveolae that mediate endothelial transcytosis. In addition, a reverse genetic study identifies the outer region of the icosahedral threefold capsid protrusions as a potential culprit for rAAV9's delayed blood clearance. These results support a model in which the delayed blood clearance of rAAV9 sustains the capacity-limited slow transvascular vector transport and plays a role in mediating robust cardiac transduction, and provide important implications in AAV capsid engineering to create new rAAV variants with more desirable properties.

Preclinical evaluation of a recombinant adeno-associated virus vector expressing human alpha-1 antitrypsin made using a recombinant herpes simplex virus production method.

Recombinant adeno-associated virus (rAAV) vectors offer promise for gene therapy of alpha-1 antitrypsin (AAT) deficiency. A toxicology study in mice evaluated intramuscular injection of an rAAV vector expressing human AAT (rAAV-CB-hAAT) produced using a herpes simplex virus (HSV) complementation system or a plasmid transfection (TFX) method at doses of 3 × 10(11) vg (1.2 × 10(13) vg/kg) for both vectors and 2 × 10(12) vg (8 × 10(13) vg/kg) for the HSV-produced vector. The HSV-produced vector had favorable in vitro characteristics in terms of purity, efficiency of transduction, and hAAT expression. There were no significant differences in clinical findings or hematology and clinical chemistry values between test article and control groups and no gross pathology findings. Histopathological examination demonstrated minimal to mild changes in skeletal muscle at the injection site, consisting of focal chronic interstitial inflammation and muscle degeneration, regeneration, and vacuolization, in vector-injected animals. At the 3 × 10(11) vg dose, serum hAAT levels were higher with the HSV-produced vector than with the TFX-produced vector. With the higher dose of HSV-produced vector, the increase in serum hAAT levels was dose-proportional in females and greater than dose-proportional in males. Vector copy numbers in blood were highest 24 hr after dosing and declined thereafter, with no detectable copies present 90 days after dosing. Antibodies to hAAT were detected in almost all vector-treated animals, and antibodies to HSV were detected in most animals that received the highest vector dose. These results support continued development of rAAV-CB-hAAT for treatment of AAT deficiency.

Reengineering a receptor footprint of adeno-associated virus enables selective and systemic gene transfer to muscle.

Reengineering the receptor footprints of adeno-associated virus (AAV) isolates may yield variants with improved properties for clinical applications. We generated a panel of synthetic AAV2 vectors by replacing a hexapeptide sequence in a previously identified heparan sulfate receptor footprint with corresponding residues from other AAV strains. This approach yielded several chimeric capsids displaying systemic tropism after intravenous administration in mice. Of particular interest, an AAV2/AAV8 chimera designated AAV2i8 displayed an altered antigenic profile, readily traversed the blood vasculature, and selectively transduced cardiac and whole-body skeletal muscle tissues with high efficiency. Unlike other AAV serotypes, which are preferentially sequestered in the liver, AAV2i8 showed markedly reduced hepatic tropism. These features of AAV2i8 suggest that it is well suited to translational studies in gene therapy of musculoskeletal disorders.

Preclinical evaluation of innate immunity to baculovirus gene therapy vectors in whole human blood.

Interactions of gene therapy vectors with human blood components upon intravenous administration have a significant effect on vector efficacy and patient safety. Here we describe methods to evaluate these interactions and their effects in whole human blood, using baculovirus vectors as a model. Opsonisation of baculovirus particles by binding of IgM and C3b was demonstrated, which is likely to be the cause of the significant blood cell-associated virus that was detected. Preventing formation of the complement C5b-9 (membrane attack) complex maintained infectivity of baculovirus particles as shown by studying the effects of two specific complement inhibitors, Compstatin and a C5a receptor antagonist. Formation of macroscopic blood clots after 4h was prevented by both complement inhibitors. Pro- and anti-inflammatory cytokines Il-1beta, IL-6, IL-8 and TNF-alpha were produced at variable levels between volunteers and complement inhibitors showed patient-specific effects on cytokine levels. Whilst both complement inhibitors could play a role in protecting patients from aggressive inflammatory reactions, only Compstatin maintained virus infectivity. We conclude that this ex vivo model, used here for the first time with infectious agents, is a valuable tool in evaluating human innate immune responses to gene therapy vectors or to predict the response of individual patients as part of a clinical trial or treatment. The use of complement inhibitors for therapeutic viruses should be considered on a patient-specific basis.

Transduction properties of adenovirus serotype 35 vectors after intravenous administration into nonhuman primates.

Adenovirus serotype 35 (Ad35) vectors have shown promise as effective gene delivery vehicles. However, the transduction profiles of Ad35 vectors in conventional mice allow only a limited estimation of transduction properties of these vectors, because the mouse analog of the subgroup B Ad receptor, CD46, is restricted to the testis. In order to assess the transduction properties of Ad35 vectors more completely, we performed transduction experiments using cynomolgus monkeys, which ubiquitously express CD46 in a pattern similar to that in humans. In vitro transduction experiments demonstrated that cultured cells from the cynomolgus monkey were efficiently transduced with Ad35 vectors. In contrast, after intravenous administration into live monkeys hardly any evidence of Ad35 vector-mediated transduction was found in any of the organs, although Ad35 vector genomes were detected in various organs. Less severe histopathological abnormalities were found in the Ad35 vector-infused monkeys than in the conventional Ad5 vector-injected monkeys. In the latter, serious tissue damage and inflammatory responses, such as hepatocyte necrosis and lymphatic hyperplasia in the colon, were induced. Both Ad35 and Ad5 vectors caused similar hematological changes (increase in CD3(+) cells, and decrease in CD16(+) cells and CD20(+) cells) in peripheral blood cells. These results should provide valuable information for the clinical application of Ad35 vectors.

Effective tumor targeted gene transfer using PEGylated adenovirus vector via systemic administration.

Conjugation of polyethylene glycol to protein or particles (PEGylation) prolongs their plasma half-lives and promotes their accumulation in tumors due to enhanced permeability and retention (EPR) effect. Although PEGylation of adenovirus vectors (Ads) is an attractive strategy to improve the in vivo kinetics of conventional Ads, the EPR effect of PEGylated Ad (PEG-Ad) had not previously been reported. In this study, we prepared PEG-Ads with PEG at various modification ratios, injected them intravenously into tumor-bearing mice, and determined the blood kinetics, viral distribution, and gene expression patterns, respectively. In addition, we conducted a cancer therapeutic study of PEG-Ad encoding tumor necrosis factor (TNF)-alpha. The plasma half-life of PEG-Ad was longer than that of unmodified-Ad, and accumulation of PEG-Ad in tumor tissue increased as the PEG modification ratio increased. In particular, PEG-Ad with about 90% modification ratio showed higher (35 times) gene expression in tumor and lower (6%) in liver, compared with values for unmodified Ad. Moreover, PEG-Ad encoding TNF-alpha demonstrated not only stronger tumor-suppressive activity but also fewer hepatotoxic side effects compared with unmodified-Ad. PEGylation of Ad achieved tumor targeting through the EPR effect, and these attributes suggest that systemic injection of PEG-Ad has great potential as an anti-tumor treatment.

Adenovirus-platelet interaction in blood causes virus sequestration to the reticuloendothelial system of the liver.

Intravenous (i.v.) delivery of recombinant adenovirus serotype 5 (Ad5) vectors for gene therapy is hindered by safety and efficacy problems. We have discovered a new pathway involved in unspecific Ad5 sequestration and degradation. After i.v. administration, Ad5 rapidly binds to circulating platelets, which causes their activation/aggregation and subsequent entrapment in liver sinusoids. Virus-platelet aggregates are taken up by Kupffer cells and degraded. Ad sequestration in organs can be reduced by platelet depletion prior to vector injection. Identification of this new sequestration mechanism and construction of vectors that avoid it could improve levels of target cell transduction at lower vector doses.

A novel method for viral gene delivery in solid tumors.

Intratumoral infusion is the most commonly used method for viral gene delivery in clinical trials for cancer treatment. However, a potential problem in this approach is that viral vectors may disseminate from tumor to normal tissues during and after the infusion. To reduce the dissemination, we developed a novel method based on a biocompatible polymer, poloxamer 407, which could significantly increase the viscosity of virus suspension when the temperature was changed from 4 degrees C to 37 degrees C. With this method, we could significantly increase transgene expression in solid tumors and reduce virus dissemination by 2 orders of magnitude after intratumoral infusion of adenoviral vectors. The mechanism of reduction was likely to be that the viscous poloxamer solution blocked convection of viral vectors in the interstitial space and the lumen of microvessels in the vicinity of the infusion site. This method has a potential to be used in the clinic for enhancing efficacy and reducing toxicity in viral gene therapy.

Persistent expression of factor VIII in vivo following nonprimate lentiviral gene transfer.

Hemophilia A is a clinically important coagulation disorder caused by the lack or abnormality of plasma coagulation factor VIII (FVIII). Gene transfer of the FVIII cDNA to hepatocytes using lentiviral vectors is a potential therapeutic approach. We investigated the efficacy of feline immunodeficiency virus (FIV)-based vectors in targeting hepatocytes and correcting FVIII deficiency in a hemophilia A mouse model. Several viral envelope glycoproteins were screened for efficient FIV vector pseudotyping and hepatocyte transduction. The GP64 glycoprotein from baculovirus Autographa californica multinuclear polyhedrosis virus pseudo-typed FIV efficiently and showed excellent hepatocyte tropism. The GP64-pseudotyped vector was stable in the presence of human or mouse complement. Inclusion of a hybrid liver-specific promoter (murine albumin enhancer/human alpha1-antitrypsin promoter) further enhanced transgene expression in hepatocytes. We generated a GP64-pseudotyped FIV vector encoding the B domain-deleted human FVIII coding region driven by the liver-specific promoter, with 2 beneficial point mutations in the A1 domain. Intravenous vector administration conferred sustained FVIII expression in hemophilia A mice for several months without the generation of anti-human FVIII antibodies and resulted in partial phenotypic correction. These findings demonstrate the utility of GP64-pseudotyped FIV lentiviral vectors for targeting hepatocytes to correct disorders associated with deficiencies of secreted proteins.

High level of stabilized angiostatin mediated by adenovirus delivery does not impair the growth of human neuroblastoma xenografts.

Human neuroblastoma (NB) is a highly heterogeneous childhood cancer secreting a high level of vascular endothelial growth factor (VEGF). Its vascularization has been clearly correlated with metastatic progression and poor outcome. Thus, molecules that target the vascular endothelium are regarded as new therapeutics of clinical interest. Angiostatin, an internal fragment of plasminogen containing the first four kringle structures, has been described as a powerful angiogenic inhibitor. We used a recombinant adenovirus encoding the human angiostatin kringle 1-3 directly fused to human serum albumin HSA (AdK3-HSA). Coupling to HSA has been previously shown to increase the in vivo half-life of this angiostatic factor, and to lead to tumor growth inhibition in the MDA-MB-231 carcinoma model. For the assessment of antiangiogenic gene therapy in the human NB IGR-N835 tumor model, 5 x 10(9) PFU of AdK3-HSA were intravenously injected in tumor-bearing athymic mice presenting either of the following experimental settings: early stage, established, and minimal residual tumors. No delay in tumor growth was observed in animals treated with AdK3-HSA as compared to those treated with the empty virus AdCO1. In early-stage tumors, kinetics of tumor occurrence and tumor growth were similar in AdK3-HSA- and AdCO1-treated animals. K3-HSA was found to be expressed at high levels (the mean value for the three experiments being 19.4+/-15.9 microg/ml) in the circulation of all animals up to 21-35 days after virus injection. In addition, IGR-N835 tumors were found to be highly vascularized and to release high amounts of angiogenic factors, in particular VEGF (665+/-370 pg/mg total protein). Thus, in spite of high circulating levels, K3-HSA may be unable to displace the NB proangiogenic switch. In this regard, a more promising target to inhibit NB angiogenesis seems to be the VEGF/VEGFR system.

Adenovirus serotype 5 fiber shaft influences in vivo gene transfer in mice.

Adenoviral vectors used in gene therapy are predominantly derived from adenovirus serotype 5 (Ad5), which infects a broad range of cells. Ad5 cell entry involves interactions with the coxsackie-adenovirus receptor (CAR) and integrins. To assess these receptors in vivo, we mutated amino acid residues in fiber and penton that are involved in receptor interaction and showed that CAR and integrins play a minor role in hepatic transduction but that integrins can influence gene delivery to other tissues. These data suggest that an alternative entry pathway exists for hepatocyte transduction in vivo that is more important than CAR or integrins. In vitro data suggest a role for heparan sulfate glycosaminoglycans (HSG) in adenovirus transduction. The role of the fiber shaft in liver uptake was examined by introducing specific amino acid changes into a putative HSG-binding motif contained within the shaft or by preparing fiber shaft chimeras between Ad5 and Ad35 fibers. Results were obtained that demonstrate that the Ad5 fiber shaft can influence gene transfer both in vitro and to the liver in vivo. These observations indicate that the currently accepted two-step entry pathway, which involves CAR and integrins, described for adenoviral infection in vitro, is not used for hepatic gene transfer in vivo. In contrast, alpha(v) integrins influence gene delivery to the lung, spleen, heart, and kidney. The detargeted vector constructs described here may provide a foundation for the development of targeted adenoviral vectors.