Actin-resistant DNAse I Expression From Oncolytic Adenovirus Enadenotucirev Enhances Its Intratumoral Spread and Reduces Tumor Growth.

Spread of oncolytic viruses through tumor tissue is essential to effective virotherapy. Interstitial matrix is thought to be a significant barrier to virus particle convection between "islands" of tumor cells. One way to address this is to encode matrix-degrading enzymes within oncolytic viruses, for secretion from infected cells. To test the hypothesis that extracellular DNA provides an important barrier, we assessed the ability of DNase to promote virus spread. Nonreplicating Ad5 vectors expressing actin-resistant DNase (aDNAse I), proteinase K (PK), hyaluronidase (rhPH20), and chondroitinase ABC (CABC) were injected into established DLD human colorectal adenocarcinoma xenografts, transcomplemented with a replicating Ad5 virus. Each enzyme improved oncolysis by the replicating adenovirus, with no evidence of tumor cells being shed into the bloodstream. aDNAse I and rhPH20 hyaluronidase were then cloned into conditionally-replicating group B adenovirus, Enadenotucirev (EnAd). EnAd encoding each enzyme showed significantly better antitumor efficacy than the parental virus, with the aDNAse I-expressing virus showing improved spread. Both DNase and hyaluronidase activity was still measurable 32 days postinfection. This is the first time that extracellular DNA has been implicated as a barrier for interstitial virus spread, and suggests that oncolytic viruses expressing aDNAse I may be promising candidates for clinical translation.

Structure-based design and optimization of potent inhibitors of the adenoviral protease.

Adenoviral infections are associated with a wide range of acute diseases, among which ocular viral conjunctivitis (EKC) and disseminated disease in immunocompromised patients. To date, no approved specific anti-adenoviral drug is available, but there is a growing need for an effective treatment of such infections. The adenoviral protease, adenain, plays a crucial role for the viral lifecycle and thus represents an attractive therapeutic target. Structure-guided design with the objective to depeptidize tetrapeptide nitrile 1 led to the novel chemotype 2. Optimization of scaffold 2 resulted in picomolar adenain inhibitors 3a and 3b. In addition, a complementary series of irreversible vinyl sulfone containing inhibitors were rationally designed, prepared and evaluated against adenoviral protease. High resolution X-ray co-crystal structures of representatives of each series proves the successful design of these inhibitors and provides an excellent basis for future medicinal chemistry optimization of these compounds.

Directed adenovirus evolution using engineered mutator viral polymerases.

Adenoviruses (Ads) are the most frequently used viruses for oncolytic and gene therapy purposes. Most Ad-based vectors have been generated through rational design. Although this led to significant vector improvements, it is often hampered by an insufficient understanding of Ad's intricate functions and interactions. Here, to evade this issue, we adopted a novel, mutator Ad polymerase-based, 'accelerated-evolution' approach that can serve as general method to generate or optimize adenoviral vectors. First, we site specifically substituted Ad polymerase residues located in either the nucleotide binding pocket or the exonuclease domain. This yielded several polymerase mutants that, while fully supportive of viral replication, increased Ad's intrinsic mutation rate. Mutator activities of these mutants were revealed by performing deep sequencing on pools of replicated viruses. The strongest identified mutators carried replacements of residues implicated in ssDNA binding at the exonuclease active site. Next, we exploited these mutators to generate the genetic diversity required for directed Ad evolution. Using this new forward genetics approach, we isolated viral mutants with improved cytolytic activity. These mutants revealed a common mutation in a splice acceptor site preceding the gene for the adenovirus death protein (ADP). Accordingly, the isolated viruses showed high and untimely expression of ADP, correlating with a severe deregulation of E3 transcript splicing.

Energy-independent helicase activity of a viral genome packaging motor.

The assembly of complex double-stranded DNA viruses includes a genome packaging step where viral DNA is translocated into the confines of a preformed procapsid shell. In most cases, the preferred packaging substrate is a linear concatemer of viral genomes linked head-to-tail. Viral terminase enzymes are responsible for both excision of an individual genome from the concatemer (DNA maturation) and translocation of the duplex into the capsid (DNA packaging). Bacteriophage λ terminase site-specifically nicks viral DNA at the cos site in a concatemer and then physically separates the nicked, annealed strands to mature the genome in preparation for packaging. Here we present biochemical studies on the so-called helicase activity of λ terminase. Previous studies reported that ATP is required for strand separation, and it has been presumed that ATP hydrolysis is required to drive the reaction. We show that ADP and nonhydrolyzable ATP analogues also support strand separation at low (micromolar) concentrations. In addition, the Escherichia coli integration host factor protein (IHF) strongly stimulates the reaction in a nucleotide-independent manner. Finally, we show that elevated concentrations of nucleotide inhibit both ATP- and IHF-stimulated strand separation by λ terminase. We present a model where nucleotide and IHF interact with the large terminase subunit and viral DNA, respectively, to engender a site-specifically bound, catalytically competent genome maturation complex. In contrast, binding of nucleotide to the low-affinity ATP binding site in the small terminase subunit mediates a conformational switch that down-regulates maturation activities and activates the DNA packaging activity of the enzyme. This affords a motor complex that binds tightly, but nonspecifically, to DNA as it translocates the duplex into the capsid shell. These studies have yielded mechanistic insight into the assembly of the maturation complex on viral DNA and its transition to a mobile packaging motor that may be common to all of the complex double-stranded DNA viruses.

Targeted expression of herpes simplex virus thymidine kinase in adenovirus-infected cells reduces virus titers upon treatment with ganciclovir in vitro.

BACKGROUND: Adenoviruses are a frequent cause of life-threatening infections in immunocompromised patients. Available therapeutics still cannot completely prevent fatal outcomes. By contrast, herpes viruses are well treatable with prodrugs such as ganciclovir (GCV), which are selectively activated in virus-infected cells by virus-encoded thymidine kinases. This effective group of prodrugs is not applicable to adenoviruses and other DNA viruses because they lack those kinases. METHODS: To render adenoviruses amenable to GCV treatment, we generated an adenoviral vector-based delivery system for targeted expression of herpes simplex virus thymidine kinase (HSV-TK) in wild-type adenovirus 5 (wt Ad5)-infected cells. HSV-TK expression was largely restricted to wt virus-infected cells by transcription of the gene from the Ad5 E4 promoter. Its activity is dependent on the adenoviral E1A gene product which is not produced by the vector but is only provided in cells infected with wt adenovirus. The anti-adenoviral effect of HSV-TK expression and concomitant treatment with GCV was assessed in vitro in four different cell lines or primary cells. RESULTS: E4 promoter-mediated HSV-TK background expression was sufficiently low to prevent cytotoxicity in the presence of low-levels GCV in cells not infected with wt Ad5. However, expression was several-fold increased in wt Ad5-infected cells and treatment with low levels of GCV efficiently inhibited wt Ad5 DNA replication. Genome copy numbers and output of infectious particles were reduced by up to > 99.99% and cell viability was greatly increased. CONCLUSIONS: We extended the concept of enzyme/prodrug therapy to adenovirus infections by selectively sensitizing adenovirus-infected cells to treatment with GCV.

The E3 ubiquitin ligase activity associated with the adenoviral E1B-55K-E4orf6 complex does not require CRM1-dependent export.

The adenovirus type 5 (Ad5) E1B-55K and E4orf6 (E1B-55K/E4orf6) proteins are multifunctional regulators of Ad5 replication, participating in many processes required for virus growth. A complex containing the two proteins mediates the degradation of cellular proteins through assembly of an E3 ubiquitin ligase and induces shutoff of host cell protein synthesis through selective nucleocytoplasmic viral late mRNA export. Both proteins shuttle between the nuclear and cytoplasmic compartments via leucine-rich nuclear export signals (NES). However, the role of their NES-dependent export in viral replication has not been established. It was initially shown that mutations in the E4orf6 NES negatively affect viral late gene expression in transfection/infection complementation assays, suggesting that E1B-55K/E4orf6-dependent viral late mRNA export involves a CRM1 export pathway. However, a different conclusion was drawn from similar studies showing that E1B-55K/E4orf6 promote late gene expression without active CRM1 or functional NES. To evaluate the role of the E1B-55K/E4orf6 NES in viral replication in the context of Ad-infected cells and in the presence of functional CRM1, we generated virus mutants carrying amino acid exchanges in the NES of either or both proteins. Phenotypic analyses revealed that mutations in the NES of E1B-55K and/or E4orf6 had no or only moderate effects on viral DNA replication, viral late protein synthesis, or viral late mRNA export. Significantly, such mutations also did not interfere with the degradation of cellular substrates, indicating that the NES of E1B-55K or E4orf6 is dispensable both for late gene expression and for the activity associated with the E3 ubiquitin ligase.

Knockdown of lung phosphodiesterase 2A attenuates alveolar inflammation and protein leak in a two-hit mouse model of acute lung injury.

Phosphodiesterase 2A (PDE2A) is stimulated by cGMP to hydrolyze cAMP, a potent endothelial barrier-protective molecule. We previously found that lung PDE2A contributed to a mouse model of ventilator-induced lung injury (VILI). The purpose of the present study was to determine the contribution of PDE2A in a two-hit mouse model of 1-day intratracheal (IT) LPS followed by 4 h of 20 ml/kg tidal volume ventilation. Compared with IT water controls, LPS alone (3.75 µg/g body wt) increased lung PDE2A mRNA and protein expression by 6 h with a persistent increase in protein through day 4 before decreasing to control levels on days 6 and 10. Similar to the PDE2A time course, the peak in bronchoalveolar lavage (BAL) neutrophils, lactate dehydrogenase (LDH), and protein concentration also occurred on day 4 post-LPS. IT LPS (1 day) and VILI caused a threefold increase in lung PDE2A and inducible nitric oxide synthase (iNOS) and a 24-fold increase in BAL neutrophilia. Compared with a control adenovirus, PDE2A knockdown with an adenovirus expressing a short hairpin RNA administered IT 3 days before LPS/VILI effectively decreased lung PDE2A expression and significantly attenuated BAL neutrophilia, LDH, protein, and chemokine levels. PDE2A knockdown also reduced lung iNOS expression by 53%, increased lung cAMP by nearly twofold, and improved survival from 47 to 100%. We conclude that in a mouse model of LPS/VILI, a synergistic increase in lung PDE2A expression increased lung iNOS and alveolar inflammation and contributed significantly to the ensuing acute lung injury.

A novel antiangiogenic effect for telomerase-specific virotherapy through host immune system.

Soluble factors in the tumor microenvironment may influence the process of angiogenesis; a process essential for the growth and progression of malignant tumors. In this study, we describe a novel antiangiogenic effect of conditional replication-selective adenovirus through the stimulation of host immune reaction. An attenuated adenovirus (OBP-301, Telomelysin), in which the human telomerase reverse transcriptase promoter element drives expression of E1 genes, could replicate in and cause selective lysis of cancer cells. Mixed lymphocyte-tumor cell culture demonstrated that OBP-301-infected cancer cells stimulated PBMC to produce IFN-gamma into the supernatants. When the supernatants were subjected to the assay of in vitro angiogenesis, the tube formation of HUVECs was inhibited more efficiently than recombinant IFN-gamma. Moreover, in vivo angiogenic assay using a membrane-diffusion chamber system s.c. transplanted in nu/nu mice showed that tumor cell-induced neovascularization was markedly reduced when the chambers contained the mixed lymphocyte-tumor cell culture supernatants. The growth of s.c. murine colon tumors in syngenic mice was significantly inhibited due to the reduced vascularity by intratumoral injection of OBP-301. The antitumor as well as antiangiogenic effects, however, were less apparent in SCID mice due to the lack of host immune responses. Our data suggest that OBP-301 seems to have antiangiogenic properties through the stimulation of host immune cells to produce endogenous antiangiogenic factors such as IFN-gamma.

Viral Ubiquitin and Ubiquitin-Like Deconjugases-Swiss Army Knives for Infection.

Posttranslational modifications of cellular proteins by covalent conjugation of ubiquitin and ubiquitin-like polypeptides regulate numerous cellular processes that are captured by viruses to promote infection, replication, and spreading. The importance of these protein modifications for the viral life cycle is underscored by the discovery that many viruses encode deconjugases that reverse their functions. The structural and functional characterization of these viral enzymes and the identification of their viral and cellular substrates is providing valuable insights into the biology of viral infections and the host's antiviral defense. Given the growing body of evidence demonstrating their key contribution to pathogenesis, the viral deconjugases are now recognized as attractive targets for the design of novel antiviral therapeutics.

In vitro analysis of cidofovir and genetically engineered TK expression as potential approaches for the intervention of ColoAd1-based treatment of cancer.

We have generated a novel oncolytic Adenovirus (Ad), ColoAd1, with significantly increased potency ( approximately 100-fold) relative to its parent viruses, Ad11p and Ad3, or to the clinically tested oncolytic Ad, ONYX-015. Although this agent has a significant increase in its therapeutic window relative to ONYX-015 or its parent viruses, its ability to intervene and control virotherapy in treated patient is an important safety consideration for a novel biological therapy, such as ColoAd1. As there are no approved treatments for Ad infections, we sought to define whether antivirals being used to experimentally treat Ad infections (cidofovir (CDV), ribavirin) had any activity against ColoAd1. In addition, we incorporated a well-described pro-drug converting enzyme, the herpes simplex virus-thymidine kinase (HSV-TK) gene, into the viral genome to test whether the expression of this enzyme directly from the virus could be exploited as a safety valve for arresting the viral infection in the presence of the pro-drug, ganciclovir. Both the antiviral drug, CDV, and the incorporation of the pro-drug-converting TK enzyme were validated as effective approaches to controlling ColoAd1 infection, and this represents an important advancement in the development of ColoAd1 as an anticancer treatment.

Development of a high-throughput assay for monitoring cAMP levels in cardiac ventricular myocytes.

G-protein-coupled receptors (GPCRs) represent the largest family of transmembrane receptors involved in cell signal transduction. Many of these GPCRs convey their pharmacological actions by regulating intracellular levels of 3',5'-cyclic adenosine monophosphate (cAMP). Although the heart expresses more than 100 GPCRs, drug agonists for approximately one third of these GPCRs have not been identified. The goal of this project was to initiate the development of a high-throughput screening assay for monitoring cAMP in the heart. Neonatal rat cardiac ventricular myocytes were isolated and cultured on coverslips (whole-cell patch clamp recording) or in 96-well plates (fluorescent imaging plate reader measurements). Cells were infected with adenovirus expressing either beta-galactosidase (AdLacZ) or a mutant cyclic nucleotide-gated (CNG) channel containing the double mutation C460W/E583M (AdCNG). Addition of 2 microM forskolin along with 100 microM 3-isobutyl-1-methylxanthine, to increase intracellular cAMP, activated a cation current in myocytes infected with the AdCNG. In myocytes loaded with the fluorescent Ca indicator Fluo-4, stimulation with forskolin, epinephrine, norepinephrine, or the beta-adrenergic receptor agonist isoproterenol increased the fluorescent signal indicative of Ca influx through the CNG channel. In conclusion, CNG channels are readily expressed in cultured cardiac myocytes and may be utilized in high-throughput screening assays of intracellular cAMP.

Modulation of telomerase promoter tumor selectivity in the context of oncolytic adenoviruses.

The telomerase RNA (hTR) and reverse transcriptase (hTERT) promoters are active in most cancer cells, but not in normal cells, and are useful for transcriptional targeting in gene therapy models. Telomerase-specific conditionally replicating adenoviruses (CRAd) are attractive vectors because they should selectively lyse tumor cells. Here, we compare CRAds, in which either the hTR or hTERT promoter controls expression of the adenovirus E1A gene. In replication-defective reporter adenoviruses, the hTR promoter was up to 57-fold stronger in cancer cells than normal cells and up to 49-fold stronger than hTERT. In normal cells, hTERT promoter activity was essentially absent. Doses of telomerase-specific CRAds between 1.8 and 28 infectious units per cell efficiently killed cancer cells, but normal cells required higher doses. However, CRAd DNA replication and E1A expression were detected in both cancer and normal cells. Overall, tumor specificity of the CRAds was limited compared with nonreplicating vectors. Surprisingly, both CRAds expressed similar E1A levels and functional behavior, despite known differentials between hTR and hTERT promoter activities, suggesting that the promoters are deregulated. Rapid amplification of cDNA ends analysis of hTR-/hTERT-E1A transcripts ruled out cryptic transcription from the vector backbone. Blocking E1A translation partially restored the hTR-/hTERT-E1A mRNA differential, evidencing feedback regulation by E1A.

Improvement of antitumor activity by gene amplification with a replicating but nondisseminating adenovirus.

Gene therapy is a promising approach for cancer treatment; however, efficacy of current vectors remains insufficient. To improve the success of suicide gene therapy, we constructed a replication-competent adenoviral vector that has its protease gene deleted and expresses bacterial cytosine deaminase fused with bacterial uracil phosphoribosyltransferase (CU). The prodrug, 5-fluorocytosine, is transformed into the highly toxic and tissue-diffusible 5-fluorouracil by CU in infected cells. This vector is incapable of producing infectious particles but is able to undergo a single round of replication, thereby increasing transgene copy number and expression. In the presence of 5-FC, compared with the first-generation vector (AdCU), the replication-competent vector, Ad(dPS)CU-IRES-E1A, was significantly more efficacious for in vitro tumor cell killing and in bystander assays, whereas 25-fold fewer viral particles were required in a three-dimensional spheroid model. For in vivo experiments, in which virus was injected into preestablished intracranial glioma xenografts, followed by 5-FC treatment, mice receiving Ad(dPS)CU-IRES-E1A had significantly smaller tumors at 35 days postinjection as well as significantly longer median survival than mice treated with the replication-deficient, protease-deleted vector [Ad(dPS)CU]. In an immunocompetent syngeneic model, Ad(dPS)CU + 5-FC-treated mice had a median survival of only 23 days, whereas Ad(dPS)CU-IRES-E1A + 5-FC-treated animals had a survival of 57.1% at 365 days. In conclusion, Ad(dPS)CU-IRES-E1A in the presence of 5-FC produces more potent tumoricidal effects than its replication-deficient counterparts.

Temporal and spatial control of the adenovirus proteinase by both a peptide and the viral DNA.

The adenovirus proteinase (AVP) uses both an 11-amino acid peptide (pVIc) and the viral DNA as cofactors to increase its catalytic rate constant 6000-fold. The crystal structure of an AVP-pVIc complex at 2.6-A resolution reveals a new protein fold of an enzyme that is the first member of a new class of cysteine proteinases, which arose via convergent evolution.

Screening the ToxCast Phase 1, Phase 2, and e1k Chemical Libraries for Inhibitors of Iodothyronine Deiodinases.

Deiodinase enzymes play an essential role in converting thyroid hormones between active and inactive forms by deiodinating the pro-hormone thyroxine (T4) to the active hormone triiodothyronine (T3) and modifying T4 and T3 to inactive forms. Chemical inhibition of deiodinase activity has been identified as an important endpoint to include in screening chemicals for thyroid hormone disruption. To address the lack of data regarding chemicals that inhibit the deiodinase enzymes, we developed robust in vitro assays that utilized human deiodinase types 1, 2, and 3 and screened over 1800 unique chemicals from the U.S. EPA's ToxCast phase 1_v2, phase 2, and e1k libraries. Initial testing at a single concentration identified 411 putative deiodinase inhibitors that produced inhibition of 20% or greater in at least 1 of the 3 deiodinase assays, including chemicals that have not previously been shown to inhibit deiodinases. Of these, 228 chemicals produced enzyme inhibition of 50% or greater; these chemicals were further tested in concentration-response to determine relative potency. Comparisons across these deiodinase assays identified 81 chemicals that produced selective inhibition, with 50% inhibition or greater of only 1 of the deiodinases. This set of 3 deiodinase inhibition assays provides a significant contribution toward expanding the limited number of in vitro assays used to identify chemicals with the potential to interfere with thyroid hormone homeostasis. In addition, these results set the groundwork for development and evaluation of structure-activity relationships for deiodinase inhibition, and inform targeted selection of chemicals for further testing to identify adverse outcomes of deiodinase inhibition.

Phorbol ester-induced apoptosis and senescence in cancer cell models.

Protein kinase C (PKC) isozymes catalyze the phosphorylation of substrates that play key roles in the control in proliferation, differentiation, and survival. Treatment of cells with phorbol esters, activators of classical and novel PKC isozymes, leads to a plethora of responses in a strict cell-type-dependent specific manner. Interestingly, a few cell models undergo apoptosis in response to phorbol ester stimulation, including androgen-dependent prostate cancer cells. This effect involves the autocrine secretion of death factors and activation of the extrinsic apoptotic cascade. We have recently found that in other models, such as lung cancer cells, phorbol esters lead to irreversible growth arrest and senescence. This chapter describes the methods we use to assess these phorbol ester responses in cancer cell models, focusing on apoptosis and senescence.

Reversal of P-glycoprotein-mediated multidrug resistance in cancer cells by the c-Jun NH2-terminal kinase.

A significant impediment to the success of cancer chemotherapy is multidrug resistance (MDR). A typical form of MDR is attributable to the overexpression of membrane transport proteins, such as P-glycoprotein, resulting in an increased drug efflux. In this study, we show that adenovirus-mediated enhancement of the c-Jun NH2-terminal kinase (JNK) reduces the level of P-glycoprotein in a dose- and time-dependent manner. Protein turnover assay shows that the decrease of P-glycoprotein is independent of its protein stability. Instead, this occurs primarily at the mRNA level, as revealed by reverse transcription-PCR analysis. We find that P-glycoprotein down-regulation requires the catalytic activity of JNK and is mediated by the c-Jun transcription factor, as either pharmacologic inhibition of JNK activity or dominant-negative suppression of c-Jun remarkably abolishes the ability of JNK to down-regulate P-glycoprotein. In addition, electrophoretic mobility shift assay reveals that adenoviral JNK increases the activator protein binding activity of the mdr1 gene in the MDR cells. We further show that the decrease of P-glycoprotein level is associated with a significant increase in intracellular drug accumulation and dramatically enhances the sensitivity of MDR cancer cells to chemotherapeutic agents. Our study provides the first direct evidence that enhancement of the JNK pathway down-regulates P-glycoprotein and reverses P-glycoprotein-mediated MDR in cancer cells.

Complete elimination of colorectal tumor xenograft by combined manganese superoxide dismutase with tumor necrosis factor-related apoptosis-inducing ligand gene virotherapy.

Manganese superoxide dismutase (MnSOD) is a latent tumor suppressor gene. To investigate the therapeutic effect of MnSOD and its mechanisms, a replication-competent recombinant adenovirus with E1B 55-kDa gene deletion (ZD55) was constructed, and human MnSOD and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) genes were inserted to form ZD55-MnSOD and ZD55-TRAIL. ZD55-MnSOD exhibited an inhibition in tumor cell growth approximately 1,000-fold greater than Ad-MnSOD. ZD55-TRAIL was shown to induce the MnSOD expression in SW620 cells. Accordingly, by the combined use of ZD55-MnSOD with ZD55-TRAIL (i.e., "dual gene virotherapy"), all established colorectal tumor xenografts were completely eliminated in nude mice. The evidence exists that the MnSOD overexpression led to a slower tumor cell growth both in vitro and in vivo as a result of apoptosis caused by MnSOD and TRAIL overexpression after adenoviral transduction. Our results showed that the production of hydrogen peroxide derived from MnSOD dismutation activated caspase-8, which might down-regulate Bcl-2 expression and induce Bax translocation to mitochondria. Subsequently, Bax translocation enhanced the release of apoptosis-initiating factor and cytochrome c. Cytochrome c finally triggered apoptosis by activating caspase-9 and caspase-3 in apoptotic cascade. Bax-mediated apoptosis seems to be dependent on caspase-8 activation because the inhibition of caspase-8 prevented Bid processing and Bax translocation. In conclusion, our dual gene virotherapy completely eliminated colorectal tumor xenografts via enhanced apoptosis, and this novel strategy points toward a new direction of cancer treatment.

Premature termination by human RNA polymerase II occurs temporally in the adenovirus major late transcriptional unit.

We have recently demonstrated pausing and premature termination of transcription by eucaryotic RNA polymerase II at specific sites in the major late transcriptional unit of adenovirus type 2 in vivo and in vitro. In further developing this as a system for studying eucaryotic termination control, we found that prematurely terminated transcripts of 175 and 120 nucleotides also occur in adenovirus type 5-infected cells. In both cases, premature termination occurs temporally, being found only during late times of infection, not at early times before DNA replication or immediately after the onset of DNA replication when late gene expression has begun (intermediate times). To examine the phenomenon of premature termination further, a temperature-sensitive mutant virus, adenovirus type 5 ts107, was used to uncouple DNA replication and transcription. DNA replication is defective in this mutant at restrictive temperatures. We found that premature termination is inducible at intermediate times by shifting from a permissive temperature to a restrictive temperature, allowing continuous transcription in the absence of continuous DNA replication. No premature termination occurs when the temperature is shifted up at early times before DNA replication. Our data suggest that premature termination of transcription is dependent on both prior synthesis of new templates and cumulative late gene transcription but does not require continuous DNA replication.

Improved distribution of small molecules and viral vectors in the murine brain using a hollow fiber catheter.

OBJECT: A hollow fiber catheter was developed to improve the distribution of drugs administered via direct infusion into the central nervous system (CNS). It is a porous catheter that significantly increases the surface area of brain tissue into which a drug is infused. METHODS: Dye was infused into the mouse brain through convection-enhanced delivery (CED) using a 28-gauge needle compared with a 3-mm-long hollow fiber catheter. To determine whether a hollow fiber catheter could increase the distribution of gene therapy vectors, a recombinant adenovirus expressing the firefly luciferase reporter was injected into the mouse striatum. Gene expression was monitored using in vivo bioluminescent imaging. To assess the distribution of gene transfer, an adenovirus expressing green fluorescent protein was injected into the striatum using a hollow fiber catheter or a needle. RESULTS: Hollow fiber catheter-mediated infusion increased the volume of brain tissue labeled with dye by 2.7 times relative to needle-mediated infusion. In vivo imaging revealed that catheter-mediated infusion of adenovirus resulted in gene expression that was 10-times greater than that mediated by a needle. The catheter appreciably increased the area of brain transduced with adenovirus relative to a needle, affecting a significant portion of the injected hemisphere. CONCLUSIONS: The miniature hollow fiber catheter used in this study significantly increased the distribution of dye and adenoviral-mediated gene transfer in the mouse brain compared with the levels reached using a 28-gauge needle. Compared with standard single-port clinical catheters, the hollow fiber catheter has the advantage of millions of nanoscale pores to increase surface area and bulk flow in the CNS. Extending the scale of the hollow fiber catheter for the large mammalian brain shows promise in increasing the distribution and efficacy of gene therapy and drug therapy using CED.