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Various methods have been developed to facilitate the generation of recombinant adenovirus vectors, and three commercially available methods have been most widely used: the homologous recombination method in E1-complement cell lines, the homologous recombination method in bacteria, and an in vitro ligation method based on simple routine plasmid construction. These methods can insert foreign genes not only into the E1 deletion region, but also into the E3 deletion region, thereby permitting the construction of a binary transgene expression system in which heterologous genes can be inserted into both the E1 and E3 regions. By modifying the latter two methods, fiber-mutant adenovirus vectors can be also constructed in order to modify vector tropism. In this paper, we review recent advances in the construction of first generation adenovirus vectors and fiber-modified adenovirus vectors.

Gene transfer using lentiviral vectors has been recently shown to be enhanced with cis-acting elements in a cell-type-dependent manner in vivo. For this reason, the study reported here was designed to modify lentiviral vectors that express lacZ, human factor IX (FIX), or human alpha1-anti-trypsin (AAT) to study the effect of different cis DNA elements on transduction efficiencies. We found that incorporation of the central polypurine tract sequence (cppt) increased transduction efficiency in vitro while increasing the transduction of non-cell-cycling hepatocytes in vivo. C57Bl/6 scid mice that were administered lentiviral vectors devoid of the cppt (2 x 10(8) transducing units (T.U.)/mouse) had 81% of their lacZ-transduced hepatocytes colabeled with the cell cycle marker 5'-bromo-2'-deoxyuridine (BrdU). In contrast, inclusion of the cppt reduced the colabeling in mouse hepatocytes by 50%. Further modifications in the lentiviral vectors were performed to enhance viral titer and gene expression. We found that the inclusion of a matrix attachment region (MAR) from immunoglobulin-kappa (Igkappa) significantly increased the transduction efficiency, as measured by transgene protein expression and proviral DNA copy number, compared with vectors without Igkappa MAR. In vitro studies using human hepatoma cells demonstrated a significant increase (two- to fourfold) in human AAT and human FIX production when the Igkappa MAR was incorporated. In vivo transduction of partially hepatectomized C57Bl/6 mice given an optimized lentiviral vector containing the cppt and Igkappa MAR (2 x 10(8) T.U./mouse) resulted in sustained therapeutic levels of serum FIX (approximately 65 ng/ml). Our study demonstrates the importance of cis-acting elements to enhancing the transduction ability of lentiviral vectors and the expression of vector transgenes.

Hepatocyte transplantation has been proposed as an alternative to whole-organ transplantation to support many forms of hepatic insufficiency. Based on a significant body of work, the technique of hepatocyte transplantation has recently moved into the clinic in order to reestablish liver function without organ transplantation or to bridge the time between whole-organ liver transplantation. In addition, hepatocyte transplantation has also been proposed as a liver-directed gene therapy for a number of inherited hepatic disorders by transplanting either freshly isolated hepatocytes or genetically altered hepatocytes. To establish a research system based on the developing technology of hepatocyte transplantation, chimeric small animal models using human hepatocytes have recently been established, which would allow the study of human hepatocyte-specific functions, such as hepatitis viral infection and replication in vivo. Various aspects related to the recent progress and existing obstacles in the area of hepatocyte transplantation are summarized in this report.

The use of recombinant adenovirus (Ad) vectors containing genetically modified capsid proteins is an attractive strategy for achieving targeted gene transfer. The HI loop of the fiber knob is a promising candidate location for the incorporation of foreign ligands for achieving this goal. However, the method of constructing an Ad vector containing a foreign ligand in the HI loop of the fiber knob has proved difficult. In this study, we developed a simple system to construct fiber-modified vectors. To do this, a vector plasmid containing a complete E1/E3-deleted Ad type 5 genome and a unique Csp45I and/or ClaI site between positions 32679 and 32680 of the Ad genome (residues threonine-546 and proline-547 of the fiber protein) was constructed. Oligonucleotides corresponding to the Arg-Gly-Asp (RGD) or Asn-Gly-Arg (NGR)-containing peptide motif (as a model) and containing a Csp45I and/or ClaI recognition site, were ligated into the Csp45I and/or ClaI-digested plasmid. The foreign transgene expression cassette was inserted into the E1 deletion site of the vector plasmid and the fiber-mutant Ad vector was produced by transfection of the PacI-digested plasmid into 293 cells. The virus containing the RGD or NGR peptide on the fiber knob was able to infect human glioma cells, which do not express coxsackievirus and adenovirus receptor (CAR), one of the Ad virus receptors, about 100-1000 times more efficient than the virus containing wild-type fiber. This suggested that the mutant virus mediated CAR-independent cell entry pathway. The simplicity of this method allows not only for easy construction of fiber-mutant Ad vectors, but also for screening of the peptides that target the vector to the desired cells and tissues.

The short duration of transgene expression remains a major obstacle for the implementation of nonviral DNA vectors in clinical gene therapy trials. Here, we demonstrate stable, long-term transgene expression in vivo by transfecting a linear DNA expression cassette (LDNA) into mouse liver. Interestingly, despite similar quantities and cellular distribution of injected DNAs in their livers, mice receiving LDNA encoding human alpha1-antitrypsin (hAAT) expressed approximately 10- to 100-fold more serum hAAT than mice injected with closed circular (cc) DNA for a period of 9 months (length of study). Furthermore, when a linear human factor IX expression cassette was delivered to factor IX-deficient mice, sustained serum concentrations of more than 4 microg/ml (80% of normal) of the human clotting factor and correction of the bleeding diathesis were obtained. Southern blot analyses indicate that, unlike ccDNA, LDNA rapidly formed large, unintegrated concatemers in vivo, suggesting that transgene persistence from plasmid-based vectors was influenced by the structure of the vector in transfected cells. No differences in transgene expression or DNA molecular structures were observed when AAV ITRs were included to flank the hAAT expression cassette in both ccDNA- and LDNA-treated animals. Linear DNA transfection provides an approach for achieving long-term expression of a transgene in vivo.

Minicircle DNA vectors allow sustained transgene expression in quiescent cells and tissues. To improve minicircle production, we genetically modified Escherichia coli to construct a producer strain that stably expresses a set of inducible minicircle-assembly enzymes, ΦC31 integrase and I-SceI homing endonuclease. This bacterial strain produces purified minicircles in a time frame and quantity similar to those of routine plasmid DNA preparation, making it feasible to use minicircles in place of plasmids in mammalian transgene expression studies.

Due to the risk of insertional mutagenesis, viral transduction has been increasingly replaced by non-viral methods to generate induced pluripotent stem (iPS) cells. One technique that has not yet been explored is the use of “minicircle” DNA, a novel compact vector that is free of bacterial DNA and capable of persistent high level expression in cells. Here, we report the use of a single minicircle vector to generate transgene-free iPS cells from adult human cells.

As with the development of therapeutics based on RNA interference and traditional messenger RNA (mRNA) targeting with antisense, it is a liver-specific target RNA—microRNA-122 (miR-122)—that has emerged as the lead candidate for a microRNA therapeutic that could have the first meaningful clinical impact. miR-122 is by far the most abundant microRNA in the liver, and possibly in the entire human microRNA repertoire.1,2 This early realization has spawned several investigations of the function of this tissue-specific microRNA. These show it to be important for liver cell identity,2,3,4 lipid metabolism,3,5 and, perhaps curiously, hepatitis C virus (HCV) replication.6 Given the sheer abundance of this microRNA and the multitude of mRNAs that are de-repressed following its inhibition,3 it is possible that more functions will emerge. Because the liver is the organ that is most amenable to the delivery of oligonucleotides following systemic administration, antisense-targeting experiments in both mice and nonhuman primates contributed quite early and very significantly to the functional definition of miR-122, especially its role in maintaining cholesterol levels3,5,7 and in HCV replication in a chimpanzee model.8 As a result of such rapid progress, clinical trials are now under way to investigate the use of miR-122 as an antisense target for the treatment of chronic HCV infection. The phase I safety trials are sponsored by Santaris Pharma and involve healthy adult volunteers.

Recombinant adeno-associated virus (rAAV) vectors stably transduce hepatocytes in experimental animals. Following portal-vein administration of rAAV vectors in vivo, single-stranded (ss) rAAV genomes become double stranded (ds), circularized, and/or concatemerized concomitant with a slow rise and, eventually, steady-state levels of transgene expression. Over time, at least some of the stabilized genomes become integrated into mouse chromosomal DNA. The mechanism(s) of formation of stable ds rAAV genomes from input ss DNA molecules has not been delineated, although second-strand synthesis and genome amplification by a rolling-circle model has been proposed. To begin to delineate a mechanism, we produced rAAV vectors in the presence of bacterial Pae R7 or Dam methyltransferase or constructed rAAV vectors labeled with different restriction enzyme recognition sites and introduced them into mouse hepatocytes in vivo. A series of molecular analyses demonstrated that second-strand synthesis and rolling-circle replication did not appear to be the major processes involved in the formation of stable ds rAAV genomes. Rather, recruitment of complementary plus and minus ss genomes and subsequent random head-to-head, head-to-tail, and tail-to-tail intermolecular joining were primarily responsible for the formation of ds vector genomes. These findings contrast with the previously described mechanism(s) of transduction based on in vitro studies. Understanding the mechanistic process responsible for vector transduction may allow the development of new strategies for improving rAAV-mediated gene transfer in vivo.

Lentiviral vectors have the potential to play an important role in hemophilia gene therapy. The present study used human immunodeficiency virus (HIV)-based lentiviral vectors containing an EF1 enhancer/promoter driving human factors VIII (hFVIII) or IX (hFIX) complementary DNA expression for portal vein injection into C57Bl/6 mice. Increasing doses of hFIX-expressing lentivirus resulted in a dose-dependent, sustained increase in serum hFIX levels up to approximately 50-60 ng/mL. Partial hepatectomy resulted in a 4- to 6-fold increase (P

We systematically compared human factor IX gene expression from a variety of plasmids containing different cis-regulatory sequences after transfection into different hepatocyte cell lines, or in vivo, after their injection into the livers of mice. Although there was a 1.5- to 2.0-fold variation in gene expression from cultured cells, a 65-fold variation was observed in the in vivo studies. We found that a plasmid containing the apolipoprotein E locus control region (HCR), human alpha1-antitrypsin (hAAT) promoter, hFIX minigene (hFIXmg) sequence including a portion of the first intron (intron A), 3'-untranslated region (3'-UTR), and a bovine growth hormone polyadenylation signal (bpA) produced the highest serum level of human factor IX, reaching 18 microg/ml (normal = 5 microg/ml) 1 day after injection. Although most of the plasmid DNAs resulted in transient gene expression, inclusion of an intron, a polyadenylation signal from either the 1.7-kb 3'-UTR or the 0.3-kb bpA, and the HCR resulted in persistent and therapeutic levels of hFIX gene expression, ranging from 0.5 to 2 microg/ml (10 to 40% of normal) for 225 days (length of experiment). These data underscore the importance of cis sequences for enhancing in vivo hepatic gene expression and reemphasize the lack of correlation of gene expression in tissue culture and in vivo studies.

Persistence of hepatocytes transplanted into the same or related species has been established. The long-term engraftment of human hepatocytes into rodents would be useful for the study of human viral hepatitis, where it might allow the species, technical and size limitations of the current animal models to be overcome. Although transgenic mice expressing the hepatitis B virus (HBV) genome produce infectious virus in their serum, the viral life cycle is not complete, in that the early stages of viral binding and entry into hepatocytes and production of an episomal transcriptional DNA template do not occur. As for hepatitis delta virus (HDV), another cause of liver disease, no effective therapy exists to eradicate infection, and it remains resistant even to recent regimens that have considerably changed the treatment of HBV (ref. 13). Here, we demonstrate long-term engraftment of primary human hepatocytes transplanted in a matrix under the kidney capsule of mice with administration of an agonistic antibody against c-Met. These mice were susceptible to HBV infection and completion of the viral life cycle. In addition, we demonstrate super-infection of the HBV-infected mice with HDV. Our results describe a new xenotransplant model that allows study of multiple aspects of human hepatitis viral infections, and may enhance studies of human liver diseases.

Pre-clinical studies in mice and haemophilic dogs have shown that introduction of an adeno-associated viral (AAV) vector encoding blood coagulation factor IX (FIX) into skeletal muscle results in sustained expression of F.IX at levels sufficient to correct the haemophilic phenotype. On the basis of these data and additional pre-clinical studies demonstrating an absence of vector-related toxicity, we initiated a clinical study of intramuscular injection of an AAV vector expressing human F.IX in adults with severe haemophilia B. The study has a dose-escalation design, and all patients have now been enrolled in the initial dose cohort (2 x 10(11) vg/kg). Assessment in the first three patients of safety and gene transfer and expression show no evidence of germline transmission of vector sequences or formation of inhibitory antibodies against F.IX. We found that the vector sequences are present in muscle by PCR and Southern-blot analyses of muscle biopsies and we demonstrated expression of F.IX by immunohistochemistry. We observed modest changes in clinical endpoints including circulating levels of F.IX and frequency of FIX protein infusion. The evidence of gene expression at low doses of vector suggests that dose calculations based on animal data may have overestimated the amount of vector required to achieve therapeutic levels in humans, and that the approach offers the possibility of converting severe haemophilia B to a milder form of the disease.

Moloney murine leukemia virus (MoMLV)-derived vectors require cell division for efficient transduction, which may be related to an inability of the viral DNA-protein complex to cross the nuclear membrane. In contrast, adenoviruses (Ad) can efficiently infect nondividing cells. This property may be due to the presence of multiple nuclear translocation signals in a number of Ad proteins, which are associated with the incoming viral genomes. Of particular interest is the Ad preterminal protein (pTP), which binds alone or in complex with the Ad polymerase to specific sequences in the Ad inverted terminal repeat. The goal of this study was to test whether coexpression of pTP with retroviral DNA carrying pTP-binding sites would facilitate nuclear import of the viral preintegration complex and transduction of quiescent cells. In preliminary experiments, we demonstrated that the karyophylic pTP can coimport plasmid DNA into the nuclei of growth-arrested cells. Retroviral transduction studies were performed with G1/S-arrested LTA cells or stationary-phase human primary fibroblasts. These studies demonstrated that pTP or pTP-Ad polymerase conferred nuclear import of retroviral DNA upon arrested cells when the retrovirus vector contained the corresponding binding motifs. However, pTP-mediated nuclear translocation of MoMLV DNA in nondividing cells was not sufficient for stable transduction. Additional cellular factors activated during S phase or DNA repair synthesis were required for efficient retroviral integration. Integration of viral DNA into the host chromosome is an essential step in the retrovirus life cycle. With their remarkably efficient ability to integrate, retrovirus vectors are an important tool in obtaining stable gene expression in vitro and in vivo (37, 65). However, the most commonly used and as yet best-characterized vectors based on mammalian C-type retroviruses cannot transduce quiescent cells, which often represent the targets for gene therapy approaches (20, 38, 43, 63). After entry of the retroviral core into the cytoplasm, the retroviral genome is reverse transcribed by using enzymatic activities associated with the incoming virion. The resulting double-stranded linear viral DNA is associated with viral proteins, forming a large nucleoprotein complex. To complete the retroviral life cycle, this preintegration complex must be translocated to the nucleus. The mechanisms of nuclear transport appear to differ among different retrovirus subfamilies and host cells (for a review, see reference 8). For Moloney murine leukemia virus (MoMLV)-derived vectors, it is believed that breakdown of the nuclear membrane during mitosis is necessary to allow access of the preintegration complex to chromosomal DNA. This is supported by two lines of evidence: (i) inhibitors, which delay the onset of mitosis, delay integration as well (3, 33, 38); and (ii) MoMLV proviruses segregate into only one daughter cell during the first mitotic division after infection, implying that integration occurs after DNA replication (19, 43). The MoMLV preintegration complex contains linear DNA associated with the CA, IN, and possibly RT and NC proteins (8). This complex with a sedimentation constant of ;160S is too large to pass through the nuclear membrane by

Recently, we demonstrated that inverted repeat sequences inserted into first-generation adenovirus (Ad) vector genomes mediate precise genomic rearrangements resulting in vector genomes devoid of all viral genes that are efficiently packaged into functional Ad capsids. As a specific application of this finding, we generated adenovirus–adeno-associated virus (AAV) hybrid vectors, first-generation Ad vectors containing AAV inverted terminal repeat sequences (ITRs) flanking a reporter gene cassette inserted into the E1 region. We hypothesized that the AAV ITRs present within the hybrid vector genome could mediate the formation of rearranged vector genomes (ΔAd.AAV) and stimulate transgene integration. We demonstrate here that ΔAd.AAV vectors are efficiently generated as by-products of first-generation adenovirus-AAV vector amplification. ΔAd.AAV genomes contain only the transgene flanked by AAV ITRs, Ad packaging signals, and Ad ITRs. ΔAd.AAV vectors can be produced at a high titer and purity. In vitro transduction properties of these deleted hybrid vectors were evaluated in direct comparison with first-generation Ad and recombinant AAV vectors (rAAVs). The ΔAd.AAV hybrid vector stably transduced cultured cells with efficiencies comparable to rAAV. Since cells transduced with ΔAd.AAV did not express cytotoxic viral proteins, hybrid viruses could be applied at very high multiplicities of infection to increase transduction rates. Southern analysis and pulsed-field gel electrophoresis suggested that ΔAd.AAV integrated randomly as head-to-tail tandems into the host cell genome. The presence of two intact AAV ITRs was crucial for the production of hybrid vectors and for transgene integration. ΔAd.AAV vectors, which are straightforward in their production, represent a promising tool for stable gene transfer in vitro and in vivo.

In the presence of a protein synthesis inhibitor, cycloheximide, tumor necrosis factor-alpha (TNF-alpha), interleukin 1-beta (IL-1beta), or lipopolysaccharide (LPS) induces human umbilical vein endothelial cells (HUVECs) to undergo apoptosis, suggesting that constitutive or inducible cytoprotective pathways are required for cell survival. We studied the correlation between nuclear factor-kappaB (NF-kappaB) activation and cell death induced by TNF-alpha, IL-1beta, or LPS. Adenovirus-mediated overexpression of a dominant-negative IkappaBalpha (inhibitor of kappaB) mutant blocked NF-kappaB activation by gel shift assay and blocked induction of vascular cell adhesion molecule-1 protein by TNF-alpha, IL-1beta, and LPS, a NF-kappaB-dependent response. In cells overexpressing the IkappaBalpha mutant, TNF-alpha induced cell death, whereas IL-1beta or LPS did not. We conclude that cell survival following TNF-alpha stimulation is NF-kappaB-dependent but that a constitutive or inducible NF-kappaB-independent pathway(s) protects IL-1beta- or LPS-treated HUVECs from cell death.

We previously developed a two-plasmid in vitro ligation method that did not require a recombination step to produce new recombinant E1- or E1/E3-deleted adenoviral vectors. In this study, we have modified the system to improve the simplicity of vector construction and, in addition, to allow for production of an E1/E4-deleted vector.

Endothelial cell death may contribute to tissue injury from ischemia. Little is known, however, about the characteristics of endothelial cell death in response to hypoxia. Using an in vitro model, we found that human umbilical vein endothelial cells were resistant to hypoxia-induced cell death with only a 2% reduction in viability at 24 h and 45% reduction in viability at 48 h. Overexpression of a mutant, IκBα, via adenoviral vector did not potentiate cell death in hypoxia, indicating that nuclear factor-κB activation was not involved in cytoprotection. Cell death in hypoxia was determined to be apoptotic by 3′ labeling of DNA using terminal deoxynucleotidyl transferase staining and reversibility of cell death with a caspase inhibitor. Exposure of endothelial cells to hypoxia did not alter levels of proapoptotic and antiapoptotic Bcl-2 family members Bax and Bcl-XL by immunoblot analysis. In contrast, changes in p53 protein levels correlated with the induction of apoptosis in hypoxic endothelial cells. Inhibition of the proteasome increased p53 protein levels and accelerated cell death in hypoxia. Overexpression of p53 by adenoviral transduction was sufficient to initiate apoptosis of normoxic endothelial cells. These data provide a framework for the study of factors regulating endothelial cell survival and death in hypoxia.

Hemophilia B, or factor IX deficiency, is an X-linked recessive disorder occurring in about 1 in 25,000 males. Affected individuals are at risk for spontaneous bleeding into many organs; treatment mainly consists of the transfusion of clotting factor concentrates prepared from human blood or recombinant sources after bleeding has started. Small- and large-animal models have been developed and/or characterized that closely mimic the human disease state. As a preclinical model for gene therapy, recombinant adeno-associated viral vectors containing the human or canine factor IX cDNAs were infused into the livers of murine and canine models of hemophilia B, respectively. There was no associated toxicity with infusion in either animal model. Constitutive expression of factor IX was observed, which resulted in the correction of the bleeding disorder over a period of over 17 months in mice. Mice with a steady-state concentration of 25% of the normal human level of factor IX had normal coagulation. In hemophilic dogs, a dose of rAAV that was approximately 1/10 per body weight that given to mice resulted in 1% of normal canine factor IX levels, the absence of inhibitors, and a sustained partial correction of the coagulation defect for at least 8 months.

Recombinant adeno-associated virus vectors have been shown to safely transduce a number of tissues in preclinical animal studies. The level of gene transfer is sufficient to successfully treat a large number of medical disorders. Moreover, the long-term persistence of the vector sequences in animals makes it likely that this vector will be useful for genetic diseases requiring life-long therapy. This review outlines the biological principles of the vector, as well as its advantages and current limitations as it relates to use in hepatic gene therapy.

Data were analyzed from 74 “Freight Mobility Interviews”—surveys conducted with key transportation executives whose products and services are shipped into New York City’s central business district (CBD). Quantitative data collected included company profiles, defined by product category; kind of transportation service; type of distribution channel; characteristics of dispatched truck trip; and time and cost for last leg of trip. Major barriers to freight mobility identified by logistics/distribution/ transportation managers were widespread congestion, theft/vandalism, inadequate docking space, and insufficient curbside parking for commercial vehicles. Recommendations to increase productivity in the CBD included off-peak and extended delivery hours, additional truck parking zones, and incentives to upgrade docking areas. Barriers to freight mobility were consistent across industry sectors. Initiatives that have the potential to increase the efficiency of urban goods movement include improved law enforcement to deter theft/vandalism, information-based improvements such as accurate signage, the use of ITS technology and management systems to actively manage curbside commercial parking zones, and improved road maintenance. The nontraditional methodology developed for collecting urban freight mobility data provides process-oriented data that reflect changing supply chain strategies of private-sector shippers and carriers.

Chromosomal integration of rAAV vectors may induce hepatocellular carcinoma in neonatally treated mice.

Integration of AAV into genomic loci has been well studied using low throughput techniques, which has revealed clear integration hotspots. Recent studies using high throughput sequencing have uncovered new genomic loci where AAV may integrate. However, the low integration frequency combined with the AAV inverted terminal repeats (ITR) makes amplification technologically challenging and prone to artifacts that may mis-identify or miss some integration sites. Our lab has utilized a modified Illumina Mate Pair sequencing technique to uncover genomic integration sites and bypass the need to amplify through the AAV ITR. We believe that this technique can accurately establish bona-fide integration sites, uncover novel integration sites, and provide further evidence for existing known hotspots.At its core, mate pair sequencing involves self-circularization of fractured genomic fragments followed by recovery and sequencing of the junction. In essence, it bridges the low throughput shuttle vector systems used in the past with high throughput sequencing. Here we have utilized biotinylated loxP linkers added to the ends of genomic fragments and at unique sites in AAV vectors followed by Cre circularization (Van Nieuwerburgh, 2012). The circularized fragments are enriched by exonuclease treatment to remove linear fragments then fractured to ~400 bp. The fragments containing the loxP junction are purified using magnetic strepavadin coated beads and Illumina sequencing adapters are added before sequencing. Using this technique, we have successfully recovered control sequences of plasmids mixed with genomic DNA. Importantly, the Cre recombination reaction allows us to accurately distinguish self-circularized fragments from cross-recombined fragments and eliminate apparent false positive integration sites. We are working on applying this technique to DNA samples from mice injected with AAV. Continued study of AAV integration will be extremely important for establishing the safety profile of AAV used in clinical applications. View Large Image | Download PowerPoint Slide

Research towards improved adeno-associated virus (AAV) delivery vectors for use in treating human liver diseases continues to be dominated by efforts to characterize potential candidate vectors in mice which often can't recapitulate either the human liver tropism/transduction profiles, or the specifics of the human immune response to these vectors. Research from our lab has shown that despite the impressive hepatocyte transduction levels by AAV-8 in mouse liver, AAV-8 does not functionally transduce human hepatocytes in xenografted humanized liver mouse models to levels of therapeutic relevance, possibly explaining the lower than expected FIX expression levels from the recent AAV-8 hemophilia clinical trial. Efforts to evolve novel capsid variants with improved human hepatocyte tropism were successful with the generation of AAV-LK03, yet limitations to clinical translation with many newly evolved serotypes remains due to high levels of pre-existing neutralizing antibodies that may cross-react and neutralize transduction. For this reason we sought to evolve improved hepatotropic variants–again in humanized liver mice–that not only retained robust human liver tropism, but also had been evolved for resistance to pre-existing neutralizing antibodies in the human population to create the ideal liver vector for use in the clinic. We utilized wild-type replicating AAV libraries of 10e5 variants via DNA shuffling of eleven different parental AAV capsids. Our libraries selectively replicate in human cells when co-administered with wild-type adenovirus, making chimeric humanized liver mice an excellent tool to allow for selection of capsids with tropism for human liver. Screens were carried out for five rounds of selection and rather than waiting for the screen to go to completion, we ended the screen with some library diversity remaining for use in a subsequent screen for neutralizing antibody evasion. All variants from round five of the human liver screen were carried forward and screened for two additional rounds for their ability to resist binding to pooled human immunoglobulins in IgG immunocapture assays. The top 100 highly selected variants were sequenced and vectorized into CAG-driven GFP preparations in seven pools based on capsid relatedness at the amino acid level. Each pool was then resubjected to additional pooled human immunoglobulin screening and the best pool was chosen. The top seven candidates from this pool were tested alongside control serotypes that represent the extremes for humoral neutralization: highest neutralization (AAV-2), lowest neutralization (AAV-DJ), as well as LK03 that falls in between. One variant from this highly selected pool has neutralization profiles on par with AAV-DJ and further characterizations are underway in humanized liver mice in vivo and various human hepatoma lines in vitro to determine if both desired characteristics were robustly retained—human liver tropism and human immune evasion.

Our goal is to develop a recombinant adeno-associated virus (AAV) vector that is both resilient to neutralization by pre-existing human antibodies, and can efficiently transduce human muscle after systemic or intramuscular administration at levels sufficient to express therapeutic quantities of human monoclonal antibodies with broad-spectrum protection against HIV for use as a prophylactic vaccine and as a treatment for HIV-infected individuals. We hypothesized that capsid library screens in human skeletal muscle cells both in vitro and in vivo would be an ideal route to accomplish these goals. We utilized wild-type replicating AAV libraries of 10e5 variants via DNA shuffling of eleven different parental AAV capsids. Our libraries selectively replicate in human cells when co-administered with wild-type adenovirus, making chimeric humanized muscle mice and human muscle cell cultures excellent tools to allow for selection of capsids with tropism for human muscle cells. Two in vivo screens are ongoing in chimeric humanized muscle mice. These screens involve transplanting immunodeficient mice with pooled primary human muscle stem cells FACS-purified from skeletal muscle biopsies from five human donors. Human muscle progenitors fuse with those from the mouse and create chimeric myofibers expressing human muscle proteins for AAV selection. AAV capsid libraries are used to serially infect xenotransplanted mice and are then replicated with either Adenovirus-19b or 36. Two in vitro screens were performed in either primary human muscle stem cells isolated and purified from patient biopsies, or differentiated human myotubes. Completed screens were carried out for six rounds of replicating selection with Adenovirus-5 and the five most highly selected variants from each screen were sequenced and vectorized into EF1a-driven GFP-luciferase dual expression vectors. We are validating these 10 highly selected variants with comparisons to parental serotypes with some known muscle tropism (AAVs 1,6,8,9), as well as establishing neutralizing antibody levels against pooled human immunoglobulins. Vectors are being tested in humanized muscle mice and various human muscle cell lines (both healthy and diseased) by four methods of administration (intra-muscular, intra-arterial, intra-venous and intra-peritoneal) at a variety of doses for robust transgene expression. Best candidates will be used to package broadly neutralizing antibodies against HIV for further pre-clinical validation as a vaccine delivery agent. More generally, we hope to provide the muscle gene therapy community with a panel of optimized capsids with enhanced tropism for numerous muscle cell types with broad potential applications.

RNAi therapeutics are emerging as a major drug discovery engine

Recombinant retroviral vectors represent an attractive means of transferring genes into the liver because they integrate in the host cell genome and result in permanent gene expression. However, efficient gene transfer is limited by the requirement of active cell division for integration. Surgical partial hepatectomy has been the traditional method of inducing hepatocellular proliferation, but this invasive approach would be difficult to justify in clinical gene therapy. As an alternative, we used a recombinant adenovirus expressing a nonsecreted form of urokinase plaminogen activator (Ad.PGKmuPA), which results in liver regeneration over a period of 8 days. When a high-titer retroviral vector was continuously infused into the portal vein of mice during this period of hepatocyte proliferation, 33.5% of hepatocytes were stably transduced. In addition, high-level expression of a secreted transgene reporter was sustained for at least 48 weeks (length of experiment). We investigated the influence of vector titer on the in vivo transduction efficiency in our system, and found that the total number of infectious retroviral particles delivered per target cell is a critical factor. The results presented here demonstrate the ability to obtain a high gene transfer efficiency and long-term gene expression in hepatocytes in vivo without the need for surgical hepatectomy. The two-vector system described here may be of clinical relevance, as the level of hepatic gene transfer achieved has potential to be curative for a large number of genetic liver diseases.

Just-in-time deliveries and lower inventories have led to more frequent deliveries of goods and services, markedly increasing urban congestion. The Goods Movement in the New York Metropolitan Area study’s goal was to develop a research methodology for capturing urban freight mobility data and to collect cost and time data on freight moving into New York City’s central business district (CBD). The methodology developed and its implementation are discussed. Problems with access and collecting data from industry executives are also addressed. In industry-sector focus groups, senior logistics executives discussed urban freight mobility issues, especially barriers to goods movement into the CBD. Barriers consistently identified in order of greatest frequency of mention from 13 focus groups were congestion, inadequate docking space, inadequate curb space for commercial vehicles, security, and excessive ticketing of high-profile companies. The Freight Mobility Interview form asked logistics/transportation/distribution managers to provide company-specific information about the following categories: transportation services and distribution channels used and related cost, time, and barriers to freight mobility. Analysis of the interview data revealed that major barriers to freight mobility identified by both shippers and carriers were consistent with those cited by focus group participants. The combined qualitative and quantitative data collected identified the processes industry uses to manage urban congestion.

In the absence of host immunity, nonintegrating, first-generation adenoviral vectors remain stable in the nucleus of quiescent transduced cells in mice. A mini-adenoviral genome (9 kb) deleted for viral E1, E2, E3, and late genes, but containing the viral inverted terminal repeats (ITRs), transgene expression cassette (human alpha 1-antitrypsin), and the viral E4 genes was equally efficient at transducing cells in vitro or in vivo as first generation, E1-deleted vectors. In contrast to a first generation vector, gene expression as well as vector DNA was short-lived in cells transduced with the deleted adenoviral genome. We demonstrate that coexpression of the adenoviral E2-preterminal protein from the vector or in trans stabilizes the mini-genome in vitro and in vivo without evidence of cellular toxicity.

Recombinant adenovirus vectors represent an efficient means of transferring genes into many different organs. The first-generation E1-deleted vector genome remains episomal and, in the absence of host immunity, persists long-term in quiescent tissues such as the liver. The mechanism(s) which allows for persistence has not been established; however, vector DNA replication may be important because replication has been shown to occur in tissue culture systems. We have utilized a site-specific methylation strategy to monitor the replicative fate of E1-deleted adenovirus vectors in vitro and in vivo. Methylation-marked adenovirus vectors were produced by the addition of a methyl group onto the N6 position of the adenine base of XhoI sites, CTCGAG, by propagation of vectors in 293 cells expressing the XhoI isoschizomer PaeR7 methyltransferase. The methylation did not affect vector production or transgene expression but did prevent cleavage by XhoI. Loss of methylation through viral replication restores XhoI cleavage and was observed by Southern analysis in a wide variety of, but not all, cell culture systems studied, including hepatoma and mouse and macaque primary hepatocyte cultures. In contrast, following liver-directed gene transfer of methylated vector in C57BL/6 mice, adenovirus vector DNA was not cleaved by XhoI and therefore did not replicate, even after a period of 3 weeks. Although replication may occur in some tissues, these results show that stabilization of the vector within the target tissue prior to clearance by host immunity is not dependent upon replication of the vector, demonstrating that the input transduced DNA genomes were the persistent molecules. This information will be useful for the design of optimal adenovirus vectors and perhaps nonviral episomal vectors for clinical gene therapy.

Haemophilia B, or factor IX deficiency, is a X-linked recessive disorder that occurs in about one in 25,000 males, and severely affected people are at risk for spontaneous bleeding into numerous organs. Bleeding can be life-threatening or lead to chronic disabilities with haemophilic arthropathy. The severity of the bleeding tendency varies among patients and is related to the concentration of functional plasma factor IX. Patients with 5-30% of the normal factor IX have mild haemophilia that may not be recognized until adulthood or after heavy trauma or surgery. Therapy for acute bleeding consists of the transfusion of clotting-factor concentrates prepared from human blood and recombinant clotting factors that are currently in clinical trials. Both recombinant retroviral and adenoviral vectors have successfully transferred factor IX cDNA into the livers of dogs with haemophilia B. Recombinant retroviral-mediated gene transfer results in persistent yet subtherapeutic concentrations of factor IX and requires the stimulation of hepatocyte replication before vector administration. Recombinant adenoviral vectors can temporarily cure the coagulation defect in the canine haemophilia B model; however, an immune response directed against viral gene products made by the vector results in toxicity and limited gene expression. The use of recombinant adeno-associated virus (rAAV) vectors is promising because the vector contains no viral genes and can transduce non-dividing cells. The efficacy of in vivo transduction of non-dividing cells has been demonstrated in a wide variety of tissues. In this report, we describe the successful transduction of the liver in vivo using r-AAV vectors delivered as a single administration to mice and demonstrate that persistent, curative concentrations of functional human factor IX can be achieved using wild-type-free and adenovirus-free rAAV vectors. This demonstrates the potential of treating haemophilia B by gene therapy at the natural site of factor IX production.

An E1a-deleted adenovirus vector constitutively express- adenovirus) and Ad/RSV-hAAT, the level and duration of ing native adenovirus E3-gp19K (Ad.RSV-gp19K) was con- serum hAAT protein were unrelated to gp19K protein structed in order to determine whether or not E3-gp19K expression. Evaluation of MHC I abundance on hepatomediated interference with antigen presentation would cytes following in vivo transduction demonstrated that result in prolonged transgene expression in vivo. Cultured recombinant adenovirus rapidly increased the abundance fibroblasts infected with Ad.RSV-gp19K produced a native of surface MHC I molecules on hepatocytes, and surface size gp19K protein and had decreased cell surface levels MHC I molecules were reduced earlier and to a greater of MHC I as shown by immunoprecipitation and flow cyto- extent following wild-type adenovirus infection compared metry. The congenic mouse strains Balb/b (H-2 b MHC I with hepatocytes transduced with control or Ad.RSVwith high gp19K affinity), Balb/k (H-2 k MHC I with no gp19K gp19K recombinant adenovirus. This difference in surface affinity), and Balb/c (H-2 d MHC I with moderate gp19K MHC I down-regulation may be related to the different proaffinity) were chosen for in vivo experiments because of moters (RSV-LTR versus the native E3 promoter), and will their range of gp19K affinities. Following transduction of be an important consideration in the development of newer mice from each strain with Ad.RSV-gp19K and Ad/RSV- generation adenovirus vectors designed to evade host hAAT (a reporter adenovirus), or Ad/RSV-cFIX (control immune responses.

Hepatocyte transplantation is a potential form of therapy for patients with genetic hepatodeficiency disorders. Unfortunately, hepatocellular transplantation has been limited because of the relatively low numbers of donor cells that can ultimately take up residence in the host liver. To give the donor cells a proliferative stimulus, a recombinant adenovirus vector that expresses a nonsecreted urokinase (urokinase-type plasminogen activator) was transduced into the livers of recipient animals before transplantation. Because urokinase production in hepatocytes causes the slow turnover of hepatocytes, 2 days after adenovirus-mediated gene transfer into the livers of recipient mice, 2 x 10(6) congenic donor cells tagged with beta-galactosidase (beta-Gal) reporter were implanted via the portal vein. As a result, on average, 8.6% of the recipient hepatocytes in the livers were derived from donor cells--a 20-fold increase compared with control animals in which no proliferative stimulus was present.

Mice lacking the enzyme fumarylacetoacetate hydrolase (FAH) have symptoms similar to humans with the disease hereditary tyrosinemia type I (HT1). FAH-deficient mice were injected with a first-generation adenoviral vector expressing the human FAH gene and followed for up to 9 months. Nontreated FAH mutant control mice died within 6 weeks from fulminant liver failure, whereas FAH adenovirus-infected animals survived until sacrifice at 2-9 months. Nine of 13 virus-treated animals developed hepatocellular cancer. Immunohistochemical analysis revealed a mosaic of FAH-deficient and FAH-positive cells in all animals and liver function tests were improved compared to controls. Even mice harvested 9 months after viral infection had50% FAH-positive cells. These results demonstrate the strong selective advantage of FAH-expressing cells in an FAH-deficient liver but also illustrate the danger of carcinomas arising from FAH-deficient hepatocytes in HT1.

Hepatitis C virus (HCV) infection is the cause of more than 90% of chronic non-A non-B hepatitis worldwide (2). It has been estimated that more than 1% of the world population is infected with HCV. Although transmission is parenteral in the majority of cases, the incidence and mechanisms of nonparenteral transmission remain uncertain. Viral infection is variable; persistent viremia occurs in about 90% of cases, chronic hepatitis occurs in 60 to 70% of cases, and 20 to 40% of infected individuals ultimately develop cirrhosis, end-stage liver disease,and/orhepatocellularcarcinoma(2).Accordingtorecent data, HCV infection is the leading indication for liver transplantation in the United States, and chronic hepatitis C is associated with more than 50% of hepatocellular carcinoma in the United States (6a). Orthotopic liver transplantation is performed in patients with end-stage liver disease, but viremia persists in 100% of patients, and ultimately, the transplanted organ becomes reinfected (11). HCV is an enveloped positive-strand RNA virus with a genome size of approximately 9.4 kb (5, 13) which encodes a single large open reading frame of ;3,010 amino acids. The singlepolyproteingeneproductundergoesproteolyticprocessing to multiple gene products, including a capsid protein, two or more envelope glycoproteins (E1 and E2), and six or more nonstructural gene products (NS2, NS3, NS4a, NS4b, NS5a, and NS5b) which are thought to be involved in viral assembly and replication, respectively (10). After the infection of hepatocytes, the genomic, positive HCV RNA strand is translated. The NS5 gene product, an RNA-dependent RNA polymerase, presumably catalyzes the synthesis of replicative, minus HCV RNAstrandswhichrepresentthetemplateforproducingmore

Myocardial infarcts heal by scar formation because there are no stem cells in myocardium, and because adult myocytes cannot divide and repopulate the wound. We sought to redirect the heart to form skeletal muscle instead of scar by transferring the myogenic determination gene, MyoD, into cardiac granulation (wound repair) tissue. A replication-defective adenovirus was constructed containing MyoD under transcriptional control of the Rous sarcoma virus long terminal repeat. The virus converted cultured cardiac fibroblasts to skeletal muscle, indicated by expression of myogenin and skeletal myosin heavy chains (MHCs). To determine if MyoD could induce muscle differentiation in vivo, we injected 2 x 10(9) or 10(10) pfu of either the MyoD or a control beta-galactosidase adenovirus into healing rat hearts, injured 1 wk previously by freeze-thaw. After receiving the lower viral dose, cardiac granulation tissue expressed MyoD mRNA and protein, but did not express myogenin or skeletal MHC. When the higher dose of virus was administered, double immunostaining showed that cells in reparative tissue expressed both myogenin and embryonic skeletal MHC. No muscle differentiation occurred after beta-galactosidase transfection. Thus, MyoD gene transfer can induce skeletal muscle differentiation in healing heart lesions. Modifications of this strategy might eventually provide new contractile tissue to repair myocardial infarcts.

Hepatitis C Virus (HCV) and other plus-strand RNA viruses typically require the generation of a small number of negative genomes (20–100× lower than the positive genomes) for replication, making the less-abundant antigenome an attractive target for RNA interference(RNAi)-based therapy. Because of the complementarity of duplex short hairpin RNA/small interfering RNA (shRNA/siRNAs) with both genomic and anti-genomic viral RNA strands, and the potential of both shRNA strands to become part of the targeting complexes, preclinical RNAi studies cannot distinguish which viral strand is actually targeted in infected cells. Here, we addressed the question whether the negative HCV genome was bioaccessible to RNAi. We first screened for the most active shRNA molecules against the most conserved regions in the HCV genome, which were then used to generate asymmetric anti-HCV shRNAs that produce biologically active RNAi specifically directed against the genomic or antigenomic HCV sequences. Using this simple but powerful and effective method to screen for shRNA strand selectivity, we demonstrate that the antigenomic strand of HCV is not a viable RNAi target during HCV replication. These findings provide new insights into HCV biology and have important implications for the design of more effective and safer antiviral RNAi strategies seeking to target HCV and other viruses with similar replicative strategies.

Prolonged warm ischemia (WI) occurring in marginal kidney donors together with reperfusion injury determines allograft survival, in which apoptosis and inflammation play crucial roles. There is no single valid biomarker, so far, to assess the degree of kidney donor injury. To define new biomarkers for detecting initial donor ischemic injury, caspase-3, caspase-7, apoptosis, inflammation, HSP70 and renal histological changes were examined in porcine kidneys subjected to 7- 15- 25- or 40-min WI, two-hour cold storage and six-hour hemoreperfusion. Caspase-3 activity was gradually increased by prolonged reperfusion, with a decrease trend against WI time. This result was verified by raised 17 kDa active caspase-3 in postreperfusion kidneys, with elevated 12 kDa active caspase-3 and lowered precursor at seven-minute WI. Active caspase-7 was also doubled by reperfusion with decreased precursor at seven-minute WI, but declined against prolonged WI. Apoptotic cells in tubular and interstitial areas were greatly increased by reperfusion at seven-minute WI, but decreased against prolonged WI. In addition, myeloperoxidase (MPO)+ cells were dramatically increased by reperfusion and presented as a bell-shape against WI time, while HSP70 was significantly increased at 7-min WI, but decreased at 40-min WI after reperfusion. In postreperfusion kidneys, tubular dilation and cell shedding were observed at 7- and 15-min WI, while tubular vacuolation and cell debris were found in tubular lumens at longer WI times. At 40-min WI, early nuclear pyknosis, tubular epithelia detachment and peri-tubular capillary dilation were detected. Furthermore, caspase-3, caspase-7, apoptosis, but not MPO+ cells or HSP70, were correlated with renal function. In conclusion, caspase-3, caspase-7 and apoptosis appear to be better biomarkers than MPO+ cells or HSP70 for assessing warm ischemic injury in donor kidneys. Hemoreperfusion activates caspase-3 and caspase-7, promotes apoptosis of damaged cells in kidneys only with limited WI, which might be beneficial to renal structural re-modeling and functional recovery.

Direct retrovirus-mediated hepatic gene transfer results in permanent gene expression; however, gene transfer requires surgical hepatectomy (to stimulate cell division) and has been inefficient. We recently used recombinant adenovirus vectors that transiently expressed urokinase from mouse hepatocytes to induce hepatocellular regeneration in place of a partial hepatectomy. The adenovirus method allowed for five-fold more efficient retrovirus transduction in vivo compared to the conventional partial hepatectomy approach. The major problem with the urokinase-mediated hepatic regeneration was the transient secretion of urokinase into the bloodstream that led to hypocoagulation. To circumvent this side-effect, the urokinase protein was modified by adding amino-terminal and carboxy-terminal endoplasmic reticulum retention signals. The recombinant urokinase molecules expressed from adenoviral vectors remained in hepatocytes, were enzymatically active, and resulted in similar rates of hepatic regeneration as found with the secreted urokinase. Modified urokinase-mediated liver regeneration was equally capable of allowing retrovirus-mediated gene transfer in vivo. Thus, the method of direct retrovirus transduction of hepatocytes becomes clinically relevant as the technology becomes safer.

Hemophilia B is caused by a deficiency of blood clotting factor IX (FIX). Previous studies have shown that the delivery of a recombinant adenoviral vector expressing canine FIX (cFIX) resulted in a complete correction of hemophilia B in FIX-deficient dogs, but that cFIX expression decreased to only about 1-2% of normal levels 3 weeks after treatment. In the present study, therapeutic levels of cFIX expression capable of producing a partial correction of hemophilia B were maintained for at least 6 months after the coadministration of the cFIX-expressing adenovirus and the immunosuppressive agent cyclosporin A (CsA). These findings support a recent report (Yang et al., 1994) that host T-cell-mediated immunity against virally transduced cells is a major contributing factor to the transient nature of adenovirus-mediated gene expression in immunocompetent animals. Although a second administration of the cFIX-expressing adenovirus 6 months after the first infusion had only a minimal effect on plasma FIX levels in a dog that had been continuously treated with CsA, the prolonged expression of the transgene indicates that immunosuppression may be applicable in attaining long-term treatment of clinically relevant disorders.

Congenital adrenal hypoplasia has been estimated to occur in approximately 1 of 12,500 births. Hyperpigmentation associated with this condition typically appears gradually during a period of months to years. We describe a newborn infant with profound hyperpigmentation in whom adrenal crisis subsequently developed as a result of congenital adrenal hypoplasia.

Retrovirus-mediated gene transfer into hepatocytes in vivo results in long-term gene expression. Limitations include the need to remove two-thirds of the liver and the relatively low frequency of gene transfer. To increase gene transfer without surgical hepatectomy, mouse hepatocytes were transduced in vivo with a recombinant adenovirus that transiently expressed urokinase, resulting in high rates of asynchronous liver regeneration. During the regenerative phase, in vivo retroviral-mediated gene transfer in hepatocytes resulted in 5- to 10-fold greater transduction efficiencies than that obtained by conventional partial hepatectomy. In 3-4 weeks, the architecture and microscopic structure of the recipient livers were normal. The two-viral system of achieving permanent transgene expression from hepatocytes in vivo offers an alternative approach to current ex vivo and in vivo gene-transfer models.

Recombinant adeno-associated viral (AAV) vectors constitute one of the most promising tools for gene transfer. While the majority of AAV transduction events are episomal, our laboratory recently exploited the vector's ability to induce homologous recombination (HR) to design promoterless vectors that utilize chromosomal homology arms flanking a ribosomal skipping P2A-therapeutic coding sequence construct to integrate sequences just upstream of the stop codon of an endogenous gene. When targeting the albumin gene in the liver, a chimeric mRNA transcript capable of producing both albumin and a second therapeutic protein is consequently created. Not only do these vectors offer the permanence of gene transfer associated with integration, but a vector lacking a promoter reduces the chance for oncogene activation from off-target vector integration. AAV-mediated HR appears to be more efficient when targeting transcriptionally active loci, yet it is unclear if transcription itself or other factors that secondarily influence transcription, such as chromatin state, are directly linked to AAV-mediated HR. We therefore set out to establish if the transcriptional rate in particular is a major determinant for this type of HR.To do this, we developed a strategy to quantify precision AAV-mediated transgene integration by exploiting an engineered locus whose transcriptional rates could be controlled by drug administration. To this end, we used lentiviral vectors to generate a series of clonal HeLa cell populations each harboring a single-copy, doxycycline-inducible genomic site expressing the human alpha 1-antitrypsin (hAAT) cDNA. Upon induction with doxycycline, transgene expression from this site increases by greater than 1,000-fold. Each clonal cell population was infected with an AAV serotype DJ vector containing homology arms to the integrated hAAT cDNA and a P2A- codon-optimized human coagulation factor IX (hFIX) cDNA or P2A-eGFP construct that would allow for multicistronic genomic expression of both peptides from a single mRNA only if HR occurred. Transgene hFIX or eGFP expression as a measure of HR is established by quantitative RT-PCR and by transgene protein measurements. By modulating the rate of transcription just prior to rAAV vector administration, we can establish if transcription from a given genomic locus influences the frequency of AAV-mediated HR. Our preliminary results suggest that transcription per se might not be the predominant factor influencing the rate of HR. Further studies will provide more insight into the mechanism of gene targeting by AAV, optimal target site selection, and potentially expand the use of AAV-mediated gene targeting for treating various genetic and acquired diseases.

Hepatitis B is a disease caused by infection with the hepatitis B virus (HBV), a small enveloped DNA virus which belongs to the Hepadnaviridae family. Even though a safe and effective vaccine is available, HBV represents the ninth most common cause of death worldwide with an estimated 350 million chronically infected individuals. HBV infection can cause cirrhosis and hepatocellular carcinoma and currently there are 7 approved drugs for the treatment of HBV infection in the USA. These drugs decrease the risk of liver damage from HBV by slowing but not eliminating viral replication in most patients, possibly related to the persistence of integrated HBV genomes. Gene therapy using recombinant adeno-associated viral (rAAV) vectors constitute a promising tool to combat HBV infection as rAAV vectors have the ability to transduce and establish long-term and stable transgene expression in liver cells. Preclinical studies have successfully utilized rAAV vectors to combat viral infections after delivering short-hairpin RNA (shRNA) expression cassettes for knocking down viral coding or host genes required for viral replication and spread. Because promiscuous targeting of host cellular genes in the majority of non-infected cells may be detrimental, specific targeting of HBV-infected cells adds a new layer of potential therapeutic targets and safety. Thus we set out to identify new AAV capsids that selectively transduce HBV-infected cells. To do this, we created new AAV shuffled capsid libraries and used these for multiple rounds of infection in HBV-infected hepatoma cells. Each library is co-infected with wild-type Adenovirus-5 (helper virus) allowing the rAAV genomes to replicate in the HBV-infected cells. Isolation of replicating rAAV over successive passages in cultured cells allows for a more stringent selection as all steps in AAV transduction must occur. We have varied the screens using various multiplicity of infections (MOIs) of the library and/or utilized a pre-clearing step using a HBV negative hepatoma cell line. Several selected capsids derived from multiple parental capsids were vectorized. One capsid (AAV-GK4) was found to transduce the infected cells approximately 6 times more robustly than uninfected cells (using an MOI of 100 and 1,000). Interestingly, the same result was observed with AAV-DJ, a variant previously selected on human hepatoma cells. Additional screens and candidates are pending further evaluation. The possibility of finding new rAAV capsids with an increased transduction efficiency and selective tropism for HBV-infected cells will provide a reagent or therapeutic that can deliver a transgene that will allow one to selectively modify or potentially kill infected but not the uninfected cells contained within the liver.

Alpha-1-antitrypsin is a relatively common genetic deficiency that results in early emphysema. The liver as the natural source of most alpha-1-antitrypsin synthesis was the target organ selected for gene replacement therapy studies. Previous work used recombinant retroviral vectors that encode the human alpha-1-antitrypsin cDNA for ex vivo and direct in vivo transduction of hepatocytes in dogs and rodents. This approach led to low levels of the human protein in the serum of recipients. In this study, recombinant adenoviral vectors that express the human alpha-1-antitrypsin cDNA under the transcriptional control of the phosphoglycerate kinase (PGK) or RSV-LTR promoters have been constructed and used for the direct transduction of mouse hepatocytes in vivo. The animals transduced with the recombinant adenoviral vectors had therapeutic serum levels of human alpha-1-antitrypsin of up to 700 μg/mL. Thus, adenovirus-mediated gene transfer of the hAAT cDNA into the liver was able to produce therapeutic serum concentrations of protein.