The non-template functions of helper virus RNAs create optimal replication conditions to enhance the proliferation of satellite RNAs.

As a type of parasitic agent, satellite RNAs (satRNAs) rely on cognate helper viruses to achieve their replication and transmission. During the infection of satRNAs, helper virus RNAs serve as templates for synthesizing viral proteins, including the replication proteins essential for satRNA replication. However, the role of non-template functions of helper virus RNAs in satRNA replication remains unexploited. Here we employed the well-studied model that is composed of cucumber mosaic virus (CMV) and its associated satRNA. In the experiments employing the CMV trans-replication system, we observed an unexpected phenomenon the replication proteins of the mild strain LS-CMV exhibited defective in supporting satRNA replication, unlike those of the severe strain Fny-CMV. Independent of translation products, all CMV genomic RNAs could enhance satRNA replication, when combined with the replication proteins of CMV. This enhancement is contingent upon the recruitment and complete replication of helper virus RNAs. Using the method developed for analyzing the satRNA recruitment, we observed a markedly distinct ability of the replication proteins from both CMV strains to recruit the positive-sense satRNA-harboring RNA3 mutant for replication. This is in agreement with the differential ability of both 1a proteins in binding satRNAs in plants. The discrepancies provide a convincing explanation for the variation of the replication proteins of both CMV strains in replicating satRNAs. Taken together, our work provides compelling evidence that the non-template functions of helper virus RNAs create an optimal replication environment to enhance satRNA proliferation.

Herpes simplex virus co-infection facilitates rolling circle replication of the adeno-associated virus genome.

Adeno-associated virus (AAV) genome replication only occurs in the presence of a co-infecting helper virus such as adenovirus type 5 (AdV5) or herpes simplex virus type 1 (HSV-1). AdV5-supported replication of the AAV genome has been described to occur in a strand-displacement rolling hairpin replication (RHR) mechanism initiated at the AAV 3' inverted terminal repeat (ITR) end. It has been assumed that the same mechanism applies to HSV-1-supported AAV genome replication. Using Southern analysis and nanopore sequencing as a novel, high-throughput approach to study viral genome replication we demonstrate the formation of double-stranded head-to-tail concatemers of AAV genomes in the presence of HSV-1, thus providing evidence for an unequivocal rolling circle replication (RCR) mechanism. This stands in contrast to the textbook model of AAV genome replication when HSV-1 is the helper virus.

Mesenchymal stromal cell delivery of oncolytic immunotherapy improves CAR-T cell antitumor activity.

The immunosuppressive tumor microenvironment (TME) is a formidable barrier to the success of adoptive cell therapies for solid tumors. Oncolytic immunotherapy with engineered adenoviruses (OAd) may disrupt the TME by infecting tumor cells, as well as surrounding stroma, to improve the functionality of tumor-directed chimeric antigen receptor (CAR)-T cells, yet efficient delivery of OAds to solid tumors has been challenging. Here we describe how mesenchymal stromal cells (MSCs) can be used to systemically deliver a binary vector containing an OAd together with a helper-dependent Ad (HDAd; combinatorial Ad vector [CAd]) that expresses interleukin-12 (IL-12) and checkpoint PD-L1 (programmed death-ligand 1) blocker. CAd-infected MSCs deliver and produce functional virus to infect and lyse lung tumor cells while stimulating CAR-T cell anti-tumor activity by release of IL-12 and PD-L1 blocker. The combination of this approach with administration of HER.2-specific CAR-T cells eliminates 3D tumor spheroids in vitro and suppresses tumor growth in two orthotopic lung cancer models in vivo. Treatment with CAd MSCs increases the overall numbers of human T cells in vivo compared to CAR-T cell only treatment and enhances their polyfunctional cytokine secretion. These studies combine the predictable targeting of CAR-T cells with the advantages of cancer cell lysis and TME disruption by systemic MSC delivery of oncolytic virotherapy: incorporation of immunostimulation by cytokine and checkpoint inhibitor production through the HDAd further enhances anti-tumor activity.

Adeno-Associated Virus Genome Interactions Important for Vector Production and Transduction.

Recombinant adeno-associated virus has emerged as one of the most promising gene therapy delivery vectors. Development of these vectors took advantage of key features of the wild-type adeno-associated virus (AAV), enabled by basic studies of the underlying biology and requirements for transcription, replication, and packaging of the viral genome. Each step in generating and utilizing viral vectors involves numerous molecular interactions that together determine the efficiency of vector production and gene delivery. Once delivered into the cell, interactions with host proteins will determine the fate of the viral genome, and these will impact the intended goal of gene delivery. Here, we provide an overview of known interactions of the AAV genome with viral and cellular proteins involved in its amplification, packaging, and expression. Further appreciation of how the AAV genome interacts with host factors will enhance how this simple virus can be harnessed for an array of vector purposes that benefit human health.

High-brightness anterograde transneuronal HSV1 H129 tracer modified using a Trojan horse-like strategy.

Neurotropic viral transsynaptic tracing is an increasingly powerful technique for dissecting the structure and function of neural circuits. Herpes simplex virus type 1 strain H129 has been widely used as an anterograde tracer. However, HSV tracers still have several shortcomings, including high toxicity, low sensitivity and non-specific retrograde labeling. Here, we aimed to construct high-brightness HSV anterograde tracers by increasing the expression of exogenous genes carried by H129 viruses. Using a Trojan horse-like strategy, a HSV/AAV (adeno-associated virus) chimaera termed H8 was generated to enhance the expression of a fluorescent marker. In vitro and in vivo assays showed that the exogenous gene was efficiently replicated and amplified by the synergism of the HSV vector and introduced AAV replication system. H8 reporting fluorescence was brighter than that of currently available H129 tracers, and H8 could be used for fast and effective anterograde tracing without additional immunostaining. These results indicated that foreign gene expression in HSV tracers could be enhanced by integrating HSV with AAV replication system. This approach may be useful as a general enhanced expression strategy for HSV-based tracing tools or gene delivery vectors.

Adenovirus vectors in hematopoietic stem cell genome editing.

Genome editing of hematopoietic stem cells (HSCs) represents a therapeutic option for a number of hematological genetic diseases, as HSCs have the potential for self-renewal and differentiation into all blood cell lineages. This review presents advances of genome editing in HSCs utilizing adenovirus vectors as delivery vehicles. We focus on capsid-modified, helper-dependent adenovirus vectors that are devoid of all viral genes and therefore exhibit an improved safety profile. We discuss HSC genome engineering for several inherited disorders and infectious diseases including hemoglobinopathies, Fanconi anemia, hemophilia, and HIV-1 infection by ex vivo and in vivo editing in transgenic mice, nonhuman primates, as well as in human CD34+ cells. Mechanisms of therapeutic gene transfer including episomal expression of designer nucleases and base editors, transposase-mediated random integration, and targeted homology-directed repair triggered integration into selected genomic safe harbor loci are also reviewed.

Helper-Dependent Adenovirus Transduces the Human and Rat Retina but Elicits an Inflammatory Reaction When Delivered Subretinally in Rats.

The identification of >100 genes causing inherited retinal degeneration and the promising results of recent gene augmentation trials have led to an increase in the number of studies investigating the preclinical efficacy of viral-mediated gene transfer. Despite success using adeno-associated viruses, many disease-causing genes, such as ABCA4 or USH2A, are too large to fit into these vectors. One option for large gene delivery is the family of integration-deficient helper-dependent adenoviruses (HDAds), which efficiently transduce postmitotic neurons. However, HDAds have been shown in other organ systems to elicit an immune response, and the immunogenicity of HDAds in the retina has not been characterized. In this study, HDAd serotype 5 (HDAd5) was found to successfully transduce rod and cone photoreceptors in ex vivo human retinal organ cultures. The ocular inflammatory response to subretinal injection of the HDAd5 was evaluated using a rat model. Subretinal injection of HDAd5 carrying cytomegalovirus promoter-driven enhanced green fluorescent protein (HDAd5-CMVp-eGFP) elicited a robust inflammatory response by 3 days postinjection. This reaction included vitreous infiltration of ionized calcium-binding adapter molecule 1 (Iba1)-positive monocytes and increased expression of the proinflammatory protein, intercellular adhesion molecule 1 (ICAM-1). By 7 days postinjection, most Iba1-positive infiltrates migrated into the neural retina and ICAM-1 expression was significantly increased compared with buffer-injected control eyes. At 14 days postinjection, Iba1-positive cells persisted in the retinas of HDAd5-injected eyes, and there was thinning of the outer nuclear layer. Subretinal injection of an empty HDAd5 virus was used to confirm that the inflammatory response was in response to the HDAd5 vector and not due to eGFP-induced overexpression cytotoxicity. Subretinal injection of lower doses of HDAd5 dampened the inflammatory response, but also eGFP expression. Despite their larger carrying capacity, further work is needed to elucidate the inflammatory pathways involved and to identify an immunomodulation paradigm sufficient for safe and effective transfer of large genes to the retina using HDAd5.

A conserved RNA structure is essential for a satellite RNA-mediated inhibition of helper virus accumulation.

As a class of parasitic, non-coding RNAs, satellite RNAs (satRNAs) have to compete with their helper virus for limited amounts of viral and/or host resources for efficient replication, by which they usually reduce viral accumulation and symptom expression. Here, we report a cucumber mosaic virus (CMV)-associated satRNA (sat-T1) that ameliorated CMV-induced symptoms, accompanied with a significant reduction in the accumulation of viral genomic RNAs 1 and 2, which encode components of the viral replicase. Intrans replication assays suggest that the reduced accumulation is the outcome of replication competition. The structural basis of sat-T1 responsible for the inhibition of viral RNA accumulation was determined to be a three-way branched secondary structure that contains two biologically important hairpins. One is indispensable for the helper virus inhibition, and the other engages in formation of a tertiary pseudoknot structure that is essential for sat-T1 survival. The secondary structure containing the pseudoknot is the first RNA element with a biological phenotype experimentally identified in CMV satRNAs, and it is structurally conserved in most CMV satRNAs. Thus, this may be a generic method for CMV satRNAs to inhibit the accumulation of the helper virus via the newly-identified RNA structure.

ICTV Virus Taxonomy Profile: Deltavirus.

Hepatitis delta virus, the only member of the only species in the genus Deltavirus, is a unique human pathogen. Its ~1.7 kb circular negative-sense RNA genome encodes a protein, hepatitis delta antigen, which occurs in two forms, small and large, both with unique functions. Hepatitis delta virus uses host RNA polymerase II to replicate via double rolling circle RNA synthesis. Newly synthesized linear RNAs are circularized after autocatalytic cleavage and ligation. Hepatitis delta virus requires the envelope of the helper virus, hepatitis B virus (family Hepadnaviridae), to produce infectious particles. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of Deltavirus which is available at www.ictv.global/report/deltavirus.

Saccharomyces paradoxus K66 Killer System Evidences Expanded Assortment of Helper and Satellite Viruses.

The Saccharomycetaceae yeast family recently became recognized for expanding of the repertoire of different dsRNA-based viruses, highlighting the need for understanding of their cross-dependence. We isolated the Saccharomyces paradoxus AML-15-66 killer strain from spontaneous fermentation of serviceberries and identified helper and satellite viruses of the family Totiviridae, which are responsible for the killing phenotype. The corresponding full dsRNA genomes of viruses have been cloned and sequenced. Sequence analysis of SpV-LA-66 identified it to be most similar to S. paradoxus LA-28 type viruses, while SpV-M66 was mostly similar to the SpV-M21 virus. Sequence and functional analysis revealed significant differences between the K66 and the K28 toxins. The structural organization of the K66 protein resembled those of the K1/K2 type toxins. The AML-15-66 strain possesses the most expressed killing property towards the K28 toxin-producing strain. A genetic screen performed on S. cerevisiae YKO library strains revealed 125 gene products important for the functioning of the S. paradoxus K66 toxin, with 85% of the discovered modulators shared with S. cerevisiae K2 or K1 toxins. Investigation of the K66 protein binding to cells and different polysaccharides implies the ß-1,6 glucans to be the primary receptors of S. paradoxus K66 toxin. For the first time, we demonstrated the coherent habitation of different types of helper and satellite viruses in a wild-type S. paradoxus strain.

Monosynaptic Tracing in Developing Circuits Using Modified Rabies Virus.

An attenuated rabies virus that expresses fluorescent protein has made it possible to analyze retrograde (presynaptic) monosynaptic connections in vivo. By combining attenuated rabies virus with a Cre-loxP based system to target cells in a subtype-specific fashion, it is possible to examine neuronal input in vivo onto any class of neuron, in development and in the mature brain. We describe here the methods to amplify deletion mutant, pseudotyped rabies virus, selectively target cells of interest using genetic and viral approaches, as well as the stereotaxic procedures required to target neuronal subtypes of interest in vivo.

Functional analysis of the N-terminal basic motif of a eukaryotic satellite RNA virus capsid protein in replication and packaging.

Efficient replication and assembly of virus particles are integral to the establishment of infection. In addition to the primary role of the capsid protein (CP) in encapsidating the RNA progeny, experimental evidence on positive sense single-stranded RNA viruses suggests that the CP also regulates RNA synthesis. Here, we demonstrate that replication of Satellite tobacco mosaic virus (STMV) is controlled by the cooperative interaction between STMV CP and the helper virus (HV) Tobacco mosaic virus (TMV) replicase. We identified that the STMV CP-HV replicase interaction requires a positively charged residue at the third position (3R) in the N-terminal 13 amino acid (aa) motif. Far-Northwestern blotting showed that STMV CP promotes binding between HV-replicase and STMV RNA. An STMV CP variant having an arginine to alanine substitution at position 3 in the N-terminal 13aa motif abolished replicase-CP binding. The N-terminal 13aa motif of the CP bearing alanine substitutions for positively charged residues located at positions 5, 7, 10 and 11 are defective in packaging full-length STMV, but can package a truncated STMV RNA lacking the 3' terminal 150 nt region. These findings provide insights into the mechanism underlying the regulation of STMV replication and packaging.

Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5.

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high packaging capacity (up to 35 kb), low immunogenicity and low toxicity. However, for many laboratories the use of HCAdV is hampered by the complicated procedure for vector genome construction and virus production. Here, a detailed protocol for efficient cloning and production of HCAdV based on the plasmid pAdFTC containing the HCAdV genome is described. The construction of HCAdV genomes is based on a cloning vector system utilizing homing endonucleases (I-CeuI and PI-SceI). Any gene of interest of up to 14 kb can be subcloned into the shuttle vector pHM5, which contains a multiple cloning site flanked by I-CeuI and PI-SceI. After I-CeuI and PI-SceI-mediated release of the transgene from the shuttle vector the transgene can be inserted into the HCAdV cloning vector pAdFTC. Because of the large size of the pAdFTC plasmid and the long recognition sites of the used enzymes associated with strong DNA binding, careful handling of the cloning fragments is needed. For virus production, the HCAdV genome is released by NotI digest and transfected into a HEK293 based producer cell line stably expressing Cre recombinase. To provide all adenoviral genes for adenovirus amplification, co-infection with a helper virus containing a packing signal flanked by loxP sites is required. Pre-amplification of the vector is performed in producer cells grown on surfaces and large-scale amplification of the vector is conducted in spinner flasks with producer cells grown in suspension. For virus purification, two ultracentrifugation steps based on cesium chloride gradients are performed followed by dialysis. Here tips, tricks and shortcuts developed over the past years working with this HCAdV vector system are presented.

A Regulatory Element Near the 3' End of the Adeno-Associated Virus rep Gene Inhibits Adenovirus Replication in cis by Means of p40 Promoter-Associated Short Transcripts.

UNLABELLED: Adeno-associated virus (AAV) has long been known to inhibit helper adenovirus (Ad) replication independently of AAV Rep protein expression. More recently, replication of Ad serotype 5 (Ad5)/AAV serotype 2 (AAV-2) hybrid vectors was shown to be inhibited incisby a sequence near the 3' end of AAVrep, termed the Rep inhibition sequence for adenoviral replication (RIS-Ad). RIS-Ad functions independently of Rep protein expression. Here we demonstrate that inhibition of adenoviral replication by RIS-Ad requires an active AAV p40 promoter and the 5' half of the intron. In addition, Ad inhibition is critically dependent on the integrity of the p40 transcription start site (TSS) leading to short p40-associated transcripts. These do not give rise to effector molecules capable of inhibiting adenoviral replication intrans, like small polypeptides or microRNAs. Our data point to an inhibitory mechanism in which RNA polymerase II (Pol II) pauses directly downstream of the p40 promoter, leading to interference of the stalled Pol II transcription complex with the adenoviral replication machinery. Whereas inhibition by RIS-Ad is mediated exclusively incis, it can be overcome by providing a replication-competent adenoviral genome intrans Moreover, the inhibitory effect of RIS-Ad is not limited to AAV-2 but could also be shown for the corresponding regions of other AAV serotypes, including AAV-5. These findings have important implications for the future generation of Ad5/AAV hybrid vectors. IMPORTANCE: Insertion of sequences from the 3' part of therepgene of adeno-associated virus (AAV) into the genome of its helper adenovirus strongly reduces adenoviral genome replication. We could show that this inhibition is mediated exclusively inciswithout the involvement oftrans-acting regulatory RNAs or polypeptides but nevertheless requires an active AAV-2 p40 promoter and p40-associated short transcripts. Our results suggest a novel inhibitory mechanism that has so far not been described for AAV and that involves stalled RNA polymerase II complexes and their interference with adenoviral DNA replication. Such a mechanism would have important implications both for the generation of adenoviral vectors expressing the AAVrepandcapgenes and for the regulation of AAV gene expression in the absence and presence of helper virus.

A Novel DNA Motif Contributes to Selective Replication of a Geminivirus-Associated Betasatellite by a Helper Virus-Encoded Replication-Related Protein.

UNLABELLED: Rolling-circle replication of single-stranded genomes of plant geminiviruses is initiated by sequence-specific DNA binding of the viral replication-related protein (Rep) to its cognate genome at the replication origin. Monopartite begomovirus-associated betasatellites can be trans replicated by both cognate and some noncognate helper viruses, but the molecular basis of replication promiscuity of betasatellites remains uncharacterized. Earlier studies showed that when tomato yellow leaf curl China virus (TYLCCNV) or tobacco curly shoot virus (TbCSV) is coinoculated with both cognate and noncognate betasatellites, the cognate betasatellite dominates over the noncognate one at the late stages of infection. In this study, we constructed reciprocal chimeric betasatellites between tomato yellow leaf curl China betasatellite and tobacco curly shoot betasatellite and assayed their competitiveness against wild-type betasatellite when coinoculated with TYLCCNV or TbCSV onto plants. We mapped a region immediately upstream of the conserved rolling-circle cruciform structure of betasatellite origin that confers the cognate Rep-mediated replication advantage over the noncognate satellite. DNase I protection and in vitro binding assays further identified a novel sequence element termed Rep-binding motif (RBM), which specifically binds to the cognate Rep protein and to the noncognate Rep, albeit at lower affinity. Furthermore, we showed that RBM-Rep binding affinity is correlated with betasatellite replication efficiency in protoplasts. Our data suggest that although strict specificity of Rep-mediated replication does not exist, betasatellites have adapted to their cognate Reps for efficient replication during coevolution. IMPORTANCE: Begomoviruses are numerous circular DNA viruses that cause devastating diseases of crops worldwide. Monopartite begomoviruses are frequently associated with betasatellites which are essential for induction of typical disease symptoms. Coexistence of two distinct betasatellites with one helper virus is rare in nature. Our previous research showed that begomoviruses can trans replicate cognate betasatellites to higher levels than noncognate ones. However, the molecular mechanisms of betasatellites selective replication remain largely unknown. We investigated the interaction between the begomovirus replication-associated protein and betasatellite DNA. We found that the replication-associated protein specifically binds to a motif in betasatellites, with higher affinity for the cognate motif than the noncognate motif. This preference for cognate motif binding determines the selective replication of betasatellites. We also demonstrated that this motif is essential for betasatellite replication. These findings shed new light on the promiscuous yet selective replication of betasatellites by helper geminiviruses.

Inhibition of HBV replication in vivo using helper-dependent adenovirus vectors to deliver antiviral RNA interference expression cassettes.

BACKGROUND: HBV is hyperendemic to southern Africa and parts of Asia, but licensed antivirals have little effect on limiting life-threatening complications of the infection. Although RNA interference (RNAi)-based gene silencing has shown therapeutic potential, difficulties with delivery of anti-HBV RNAi effectors remain an obstacle to their clinical use. To address concerns about the transient nature of transgene expression and toxicity resulting from immunostimulation by recombinant adenovirus vectors (Ads), utility of RNAi-activating anti-HBV helper-dependent (HD) Ads were assessed in this study. METHODS: Following intravenous administration of 5×10(9) unmodified or pegylated HD Ad infectious particles to HBV transgenic mice, HBV viral loads and serum HBV surface antigen levels were monitored for 12 weeks. Immunostimulation of HD Ads was assessed by measuring inflammatory cytokines, hepatic function and immune response to the co-delivered LacZ reporter gene. RESULTS: Unmodified and pegylated HD Ads transduced 80-90% of hepatocytes and expressed short hairpin RNAs (shRNAs) were processed to generate intended HBV-targeting guides. Markers of HBV replication were decreased by approximately 95% and silencing was sustained for 8 weeks. Unmodified HD Ads induced release of proinflammatory cytokines and there was evidence of an adaptive immune response to ß-galactosidase. However the HD Ad-induced innate immune response was minimal in preparations that were enriched with infectious particles. CONCLUSIONS: HD Ads have potential utility for delivery of therapeutic HBV-silencing sequences and alterations of these vectors to attenuate their immune responses may further improve their efficacy.

Factors influencing helper-independent adeno-associated virus replication.

The inability of Adeno-Associated Virus (AAV) to replicate on its own is a strong argument in favor of the use of recombinant AAV vectors for in vivo gene transfer. However, some previous studies suggested that AAV may become replication competent in cells exposed to a genotoxic stress even in the absence of co-infection with a helper virus. To comprehensively explore this phenomenon, we examined AAV genome replication in several human cell lines exposed to different genotoxic conditions. We found that all treatments induced only negligible levels of AAV replication never exceeding ten fold above background. Further investigation indicated that induction of helper-independent AAV replication relied on the synergistic contribution of several extrinsic factors linked to the origin of the cell line and the quality of the AAV preparation. These results further support the notion that helper independent AAV replication cannot occur at significant levels in vivo.

Influence of loxP insertion upstream of the cis-acting packaging domain on adenovirus packaging efficiency.

First-generation AdV enables efficient gene transduction, although its immunogenicity is an important problem in vivo. Helper-dependent AdV (HD-AdV) is one possible solution to this problem. The construction of HD-AdV requires a helper virus, in which the viral packaging domain is flanked by two inserted loxP to hamper its packaging in Cre-expressing 293 cells. Here, we constructed 19L viruses containing loxP at 191 nt from the left end of the genome upstream of the packaging domain, 15L viruses bearing loxP at 143 nt, and a control ΔL virus lacking loxP at these positions. The 19L position is used worldwide, and the 15L position has been reported to result in a lower titer than that of 19L. When the titers were compared for six pairs of 19L and 15L AdV, the 19L AdV produced titers similar to, or sometimes lower than, the 15L and ΔL AdV, unlike the results of previous reports. We next chose one pair of 15L and 19L AdV that produced titers similar to that of ΔL and a competitor AdV lacking loxP for use in a competition assay. When a small amount of the competitor AdV was co-infected, both the 15L and the 19L AdV, but not ΔL, gradually became minority components during subsequent viral passages. Therefore, the loxP insertions at 143 nt and 191 nt decreased the viral packaging efficiency.

Silencing suppressor activity of a begomovirus DNA ß encoded protein and its effect on heterologous helper virus replication.

DNA ß satellites are circular single-stranded molecules associated with some monopartite begomoviruses in the family Geminiviridae. They co-infect with their helper viruses to induce severe disease in economically important crops. The ßC1 protein encoded by DNA ß is a pathogenicity determinant and has been reported to suppress post-transcriptional gene silencing (PTGS). The ßC1 proteins from various DNA ß molecules show low levels of amino acid sequence conservation. We show here that the ßC1 from DNA ß associated with Cotton leaf curl Multan virus (CLCuMV) is a suppressor of systemic PTGS. When this DNA ß satellite co-inoculated with a heterologous helper virus, Tomato leaf curl virus (ToLCV), reduced the level of ToLCV siRNA and this was associated with a higher level of virus accumulation in infected tobacco plants. This may be a mechanism by which ßC1 protects a heterologous virus from host gene silencing.

Helper-dependent adenovirus is superior to first-generation adenovirus for expressing transgenes in atherosclerosis-prone arteries.

OBJECTIVE: Vascular gene transfer is a powerful tool for investigating and treating vascular diseases; however, its utility is limited by brevity of transgene expression and vector-associated inflammation. Helper-dependent adenovirus (HDAd), an advanced-generation adenovirus that lacks all viral genes, is superior to first-generation adenovirus (FGAd) in normal rabbit arteries. We compared HDAd to FGAd in arteries of cholesterol-fed rabbits, a model of early atherogenesis in which transgene expression might be decreased, and inflammation increased. METHODS AND RESULTS: Carotid arteries of chow- and cholesterol-fed rabbits were infused with FGAd, HDAd, or medium. HDAd expressed a transgene at least as well in arteries of cholesterol-fed rabbits as in arteries of chow-fed rabbits and expressed more durably than FGAd. In arteries of cholesterol-fed rabbits, HDAd stimulated less intimal growth, lipid deposition, and inflammation than FGAd. Neither vector affected phenylephrine-induced contraction or nitroprusside-mediated relaxation; however, both vectors decreased maximal acetylcholine-stimulated vasorelaxation. The relative absence of intimal growth in HDAd arteries could interfere with the utility of this model for testing atheroprotective genes; however, both coinfusion of FGAd and extension of cholesterol feeding yielded larger intimal lesions, on which atheroprotective genes could be tested. CONCLUSION: HDAd is superior to FGAd for expression of transgenes in atherosclerosis-prone arteries.