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The combination of controlled release technology and targeted drug delivery has become a promising strategy for cancer therapy. In this study, cell-nanoparticle hybrid vector was constructed using mesenchymal stem cells as the targeting cellular carrier and biotinylated chitosan polymer nanoparticles as the drug depot. Drug-loaded nanoparticles (hydrodynamic size =377.0 ± 14.6 nm and zeta potential = 9.6 ± 1.9 mV) were prepared by encapsulating hydrophobic model drug curcumin into biotinylated chitosan polymer. The biotin-modified nanoparticles were anchored on biotinylated mesenchymal stem cells surface by biotin-avidin binding, achieving an upload of 54.73 ± 3.95 pg/cell. The anchorage of nanoparticles on mesenchymal stem cells had no effect on their viability and homing property. Biotin-avidin binding lasted over 48 h, which could be sufficient for cell-directed tumor-tropic delivery. The in vitro and in vivo anti-tumor results advocate that cell-nanoparticle hybrid vector could prove beneficial in pulmonary melanoma metastasis therapy.

Carriers of genetic material are divided into vectors of viral and non-viral origin. Viral carriers are already successfully used in experimental gene therapies, but despite advantages such as their high transfection efficiency and the wide knowledge of their practical potential, the remaining disadvantages, namely, their low capacity and complex manufacturing process, based on biological systems, are major limitations prior to their broad implementation in the clinical setting. The application of non-viral carriers in gene therapy is one of the available approaches. Poly(amidoamine) (PAMAM) dendrimers are repetitively branched, three-dimensional molecules, made of amide and amine subunits, possessing unique physiochemical properties. Surface and internal modifications improve their physicochemical properties, enabling the increase in cellular specificity and transfection efficiency and a reduction in cytotoxicity toward healthy cells. During the last 10 years of research on PAMAM dendrimers, three modification strategies have commonly been used: (1) surface modification with functional groups; (2) hybrid vector formation; (3) creation of supramolecular self-assemblies. This review describes and summarizes recent studies exploring the development of PAMAM dendrimers in anticancer gene therapies, evaluating the advantages and disadvantages of the modification approaches and the nanomedicine regulatory issues preventing their translation into the clinical setting, and highlighting important areas for further development and possible steps that seem promising in terms of development of PAMAM as a carrier of genetic material.

Background We developed a non-viral vector, a combination of HIV-1 Tat peptide modified with histidine and cysteine (mTat) and polyethylenimine, jetPEI (PEI), displaying the high efficiency of plasmid DNA transfection with little toxicity. Since the highest efficiency of INTERFERin (INT), a cationic amphiphilic lipid-based reagent, for small interfering RNA (siRNA) transfection among six commercial reagents was shown, we hypothesized that combining mTat/PEI with INT would improve transfection efficiency of siRNA delivery. To elucidate the efficacy of the hybrid vector for siRNA silencing, β-actin expression was measured after siRNA β-actin was transfected with mTat/PEI/INT or other vectors in HSC-3 human oral squamous carcinoma cells. Results mTat/PEI/INT/siRNA produced significant improvement in transfection efficiency with little cytotoxicity compared to other vectors and achieved ≈ 100% knockdown of β-actin expression compared to non-treated cells. The electric charge of mTat/PEI/INT/siRNA was significantly higher than INT/siRNA. The particle size of mTat/PEI/INT/siRNA was significantly smaller than INT/siRNA. Filipin III and β-cyclodextrin, an inhibitor of caveolae-mediated endocytosis, significantly inhibited mTat/PEI/INT/siRNA transfection, while chlorpromazine, an inhibitor of clathrin-mediated endocytosis, did not inhibit mTat/PEI/INT/siRNA transfection. Furthermore, the transfection efficiency of mTat/PEI/INT at 4 °C was significantly lower than 37 °C. Conclusions These findings demonstrated the feasibility of using mTat/PEI/INT as a potentially attractive non-viral vector for siRNA delivery.

Oncolytic adenovirus (Ad) holds great promise as a potential gene therapy for cancer. However, intravenously administered Ad may encounter difficulties due to unfavorable host responses, non-specific interactions, and the heterogeneity of the tumor cell population. As an approach to combine the advantages of oncolytic Ad and synthetic polymers and to address the associated difficulties, Ad was physically complexed with a pH-sensitive block copolymer, methoxy poly(ethylene glycol)- b -poly( l -histidine) (mPEG- b -pHis). The in vitro transduction efficiency at an acidic extracellular pH was remarkably enhanced in cancer cells when treated with the Ad expressing green fluorescent protein (GFP) coated with mPEG- b -pHis (c-dE1/GFP) as compared to that of naked Ad (n-dE1/GFP). Time-lapse total internal reflection fluorescence microscopic imaging revealed a significantly enhanced cellular uptake rate of c-dE1/GFP at acidic tumor pH when compared with that at neutral pH or naked cognate Ad (n-dE1/GFP). In addition, c-dE1/GFP remained relatively stable in human serum-containing media, and considerably reduced both the innate and adaptive immune response against Ad. Moreover, the therapeutic efficacy and survival benefit of mPEG- b -pHis-complexed oncolytic Ad (c-H5mT/Luc) by systemic treatment was significantly enhanced compared to that with naked oncolytic Ad (n-H5mT/Luc) in both coxsackie and adenovirus receptor-positive and -negative tumors. Whole-body bioluminescence imaging showed 7.3-fold higher luciferase expression at the tumor site and 23.0-fold less luciferase expression in liver tissue for c-H5mT/Luc relative to that for naked oncolytic Ad (n-H5mT/Luc). Considering the heterogeneity of tumor tissue, these results are important for guiding the development of more potent and specific treatment of devastating metastatic cancers using this viral system. Statement of significance Although adenoviral systems have shown considerable promise and undergone extensive evaluation attempts to specifically target Ad vectors to cancer cells have met limited success. This shortcoming is due to the strong immune response stimulated by Ad and the hepatotoxicity of the viral particles. To overcome restricted vector issues, we generated Ad/mPEG- b -pHis for tumor microenvironment-targeting hybrid vector systems, an oncolytic Ad coated with a pH-responsive polymer, mPEG- b -pHis. The Ad/mPEG- b -pHis exhibited pH-dependent transduction efficiency and cancer-cell killing effects. Moreover, systemic administration of oncolytic Ad/mPEG- b -pHis led to marked suppression of tumor growth and tumor-specific viral replication. Ad successfully avoided the innate and adaptive immune responses and liver accumulation with the help of mPEG- b -pHis on its surface.

X-linked agammaglobulinemia (XLA) is one of the most common humoral immunodeficiencies, which is caused by mutations in Bruton's tyrosine kinase (BTK) gene. To examine the possibility of using gene therapy for XLA, we constructed a helper-dependent adenovirus/adeno-associated virus BTK targeting vector (HD-Ad.AAV BTK vector) composed of a genomic sequence containing BTK exons 6-19 and a green fluorescence protein-hygromycin cassette driven by a cytomegalovirus promoter. We first used NALM-6, a human male pre-B acute lymphoblastic leukemia cell line, as a recipient to measure the efficiency of gene targeting by homologous recombination. We identified 10 clones with the homologous recombination of the BTK gene among 107 hygromycin-resistant stable clones isolated from two independent experiments. We next used cord blood CD34⁺ cells as the recipient cells for the gene targeting. We isolated colonies grown in medium containing cytokines and hygromycin. We found that the targeting of the BTK gene occurred in four of the 755 hygromycin-resistant colonies. Importantly, the gene targeting was also observed in CD19⁺ lymphoid progenitor cells that were differentiated from the homologous recombinant CD34⁺ cells during growth in selection media. Our study shows the potential for the BTK gene therapy using the HD-Ad.AAV BTK vector via homologous recombination in hematopoietic stem cells.

Baculovirus is a promising vector for transducing numerous types of mammalian cells. We have developed hybrid baculovirus vectors and protocols for the efficient transduction of a variety of cell lines, primary cells and stem cells, including bone marrow-derived mesenchymal stem cells (BMSCs) and adipose-derived stem cells (ASCs). The hybrid vector enables intracellular minicircle formation and prolongs transgene expression. The advantages of this transduction protocol are that baculovirus supernatant alone needs to be added to cells growing in medium, and transduction occurs after only 4-6 h of incubation at room temperature (25 °C) with gentle shaking. The entire procedure, from virus generation to transduction, can be completed within 4 weeks. Compared with other transduction procedures, this protocol is simple and can confer efficiencies >95% for many cell types. This protocol has potential applications in tissue regeneration, as transduced cells continue to express transgenes after implantation. For example, transduction of rabbit ASCs (rASCs) with growth factor-encoding hybrid baculovirus vectors, as described as an example application in this protocol, enables robust and sustained growth factor expression, stimulates stem cell differentiation and augments tissue regeneration after implantation.

Vector delivery is still a bottleneck for gene therapy. To overcome some disadvantages of adenoviral and retroviral vectors, we developed a hybrid vector. This hybrid vector, AdLTR-luc, was created by adding two elements from Moloney murine leukemia virus (MoMLV) flanking the luciferase cDNA into an E1/E3-deleted, replication deficient serotype 5 adenovirus vector (Zheng et al., Nature Biotechnol, 2000), and demonstrated that the MoMLV element upstream of the luciferase cDNA was broken during the integration event. The purpose of the current study was to determine if the MoMLV element downstream of the luciferase cDNA was also broken when integration occurred. We used the same A5 cell clones (#10 and 11) from the earlier the paper along with restriction endonuclease digestions, plus Southern hybridization, and PCR. Southern hybridization indicated that the luciferase cDNA was intact in the cloned cells. Results from Xho I and Sal I digestions showed that integration occurred in cloned cells. Southern hybridizations after Nco I digestion suggested that there was a break in both MoMLV elements, upstream and downstream of the luciferase cDNA. After DNA digestion with Not I, hybridization analyses indicated that the MoMLV upstream element was broken during integration. Digestion of genomic DNA with either Xba I/Kpn I, Bam HI/Sac I, or Bam HI/Nco I demonstrated that the MoMLV downstream element was also broken during integration. A PCR assay was unable to amplify the junctional region between the downstream MoMLV element and the adenoviral E2B gene, consistent with a break in that element. Although AdLTR-luc integration is atypical (Zheng et al., Nature Biotechnol, 2000), the present results suggest that both MoMLV elements have important roles in this event.

Background A baculovirus vector is capable of efficiently transducing many nondiving and diving cell types. However, the potential of baculovirus is restricted for many gene delivery applications as a result of the transient gene expression that it mediates. The plasmid-based Sleeping Beauty (SB) transposon system integrates transgenes into target cell genome efficiently with a genomic integration pattern that is generally considered safer than the integration of many other integrating vectors; yet efficient delivery of therapeutic genes into cells of target tissues in vivo is a major challenge for nonviral gene therapy. In the present study, SB was introduced into baculovirus to obtain novel hybrid vectors that would combine the best features of the two vector systems (i.e. effective gene delivery and efficient integration into the genome), thus circumventing the major limitations of these vectors. Methods We constructed and optimized SB-baculovirus hybrid vectors that bear either SB100x transposase or SB transposon in the forward or reverse orientations with respect to the viral backbone The functionality of the novel hybrid vectors was investigated in cell cultures and in a proof-of-concept study in the mouse eye. Results The hybrid vectors showed high and sustained transgene expression that remained stable and demonstrated no signs of decline during the 2 months follow-up in vitro. These results were verified in the mouse eye where persistent transgene expression was detected two months after intravitreal injection. Conclusions Our results confirm that (i) SB-baculovirus hybrid vectors mediate long-term gene expression in vitro and in vivo, and (ii) the hybrid vectors are potential new tools for the treatment of ocular diseases. Copyright © 2014 John Wiley & Sons, Ltd.

Polyethylenimine (PEI) was used recently with murine leukemia virus-like particles (MLV-VLPs) to produce a hybrid vector that possesses advantages over the native virus; the transduction efficiency of this vector, however, was less than the transduction efficiency of the native virus. The cause of the reduced efficiency was hypothesized to be related to the involvement of proteins in PEI/MLV-VLP complex formation and overall complex size. To test the hypothesis and potentially improve the efficiency of the hybrid vector, ultracentrifugation and size exclusion chromatography were used to purify MLV-VLP and to study the effect of proteins in cell culture medium on complex formation. Based on dynamic light scattering and electron microscopy, complexes formed from the purified MLV-VLPs were smaller, but surprisingly, less efficient than complexes formed from unpurified MLV-VLPs. The addition of protein to purified MLV-VLPs showed that the initial efficiency could be restored and that the purification strategy was not inactivating the MLV-VLPs. Further, by optimizing the amount of protein added to the purified MLV-VLPs, the level of transduction by PEI/MLV-VLP improved 1.6-fold. Particle characterization showed a correlation between the size of the PEI/MLV-VLP complex and the transduction efficiency, which is likely a result of greater sedimentation and cell contact during in vitro studies.

Background Genetic treatments of chronic arthritic conditions are essentially dependent on safe and efficient vector systems. To combine features of the efficient transduction of adenovirus vectors with the advantage of stable integration into the host cell genome of apathogenic prototype foamy virus vectors, hybrid vectors (FAD) have been established. In the present study, we have generated and investigated the use of safe FAD vectors for direct gene delivery to joints. Methods We generated recombinant FAD encoding enhanced green fluorescent protein (EGFP) or human interleukin 1 receptor antagonist protein (IL1RA) cDNA, and explored their transgene expression profile, as well as the bioactivity of the IL1RA transgene in vitro. The feasibility of IL1RA gene delivery to articular tissues was investigated in a pilot study employing direct FAD injections to the knee joints of Wistar rats. Results FAD vectors efficiently transduced human or rat fibroblasts with EGFP or IL1RA transgene in vitro. Levels of IL1RA transgene expression were high, stable and functional in vitro. Transduced synovial fibroblasts and high levels of IL1RA protein (10–35 ng/ml) could be detected in vivo in the synovium of Wistar rats 3–5 days after injection of FAD vectors to the knee joints. Conclusions Our results indicate that FAD vectors are capable of efficient in vivo gene transfer to synovium and merit further investigation as a means of providing efficient and long-term intra-articular transgene expression for treatment of the arthritides. Copyright © 2013 John Wiley & Sons, Ltd.

Inflammatory breast cancer (IBC) is a subtype of breast cancer that has a frequent association with metastatic disease and a poorer prognosis than comparative non-inflammatory breast cancers. While IBC is now considered a distinct subclass of breast cancer, the lack of molecular characterization, both at the genomic and proteomic levels, has hampered the development of rationalized and targeted therapies. Inherent receptor/ligand interactions that can occur on the surface of tumor cells can act as a dynamic molecular address that can enable targeted delivery of drugs and imaging agents to tumors. We hypothesize that such molecular addresses within IBC can be exploited for ligand-based imaging and early detection of disease sites. To this end, it is our goal to generate targeted imaging and therapeutic agents by combining ligand-directed targeting with efficient transduction of IBC cells by hybrid gene delivery vectors. Our strategy utilizes a hybrid vector with genomic elements from adeno-associated virus (AAV) and an M13-derived phage. Ligand-targeted, AAV/phage (AAVP) chimeras can display tumor-homing peptides that mediate selective internalization of viral particles through specific ligand-receptor interactions in vitro and in vivo. Such targeted vehicles are suited for the delivery of different reporter genes that can be used for imaging, diagnosis and therapy of breast cancer As a part of our ongoing studies we have identified, characterized and evaluated a peptide (WIFPWIQL, amino acid sequence) that can target GRP78, a stress-response protein that is expressed in IBC tumors and elevated during metastatic progression. Indeed, we found that this GRP78-targeting peptide can bind to, and internalize within IBC cells. As a result, we sought to characterize the ability of this peptide to mediate the delivery of fluorescent-based compounds and toxic moieties in preclinical models of IBC and breast cancer metastasis. Using amine-based chemical coupling, we conjugated near-infrared dyes on both WIFPWIQL-phage and on a WIFPWIQL-peptide engrafted antibody. When these fluorescent construct were administered into mice bearing IBC or IBC-like tumors, we could visualize tumor-specific targeting of the vectors in vivo. To demonstrate efficacy of GRP78-targeted therapeutics, we conjugated the tumor-homing, GRP78 ligand to a cell-death inducing domain (creating a compound called BMTP-78, βone metastasis targeting peptide-78), which can selectively kill cells upon internalization. We show here that BMTP78 therapy in mice with established GRP78-positive tumors, but not matched GRP78-negative tumors, could effectively reduce tumor growth and metastatic burden. Finally, we demonstrate a WIFPWIQL-AAVP construct that expresses a suicide gene (HSVtk) under the control of either CMV or GRP78 promoter, could sensitize IBC tumor xenografts to the pro-drug ganciclovir. Collectively, our results demonstrate an in vivo receptor/ligand system that has the potential for imaging and therapeutic targeting of IBC and aggressive breast tumors. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-10-09.

An improved adenoviral-based gene delivery vector was developed by complexing adenovirus (Ad) with a biocompatible, grafted copolymer PEG-g-PEI composed of polyethylene glycol (PEG) and polyethylenimine (PEI). Although an Ad-based gene vector is considered relatively safe, its native tropism, tendency to elicit an immune response, and susceptibility to inactivating antibodies makes the virus less than ideal. The goal of the current study was to determine whether Ad could be complexed with a PEG-g-PEI copolymer that would enable the virus to transduce cells lacking the Ad receptor, while avoiding the issues commonly associated with PEI. A copolymer library was synthesized using 2 kDa PEG and either linear or branched PEI (25 kDa) with a PEG to PEI grafting ratio of 10, 20, or 30. The results of the study indicate that PEG-g-PEI/Ad complexes are indeed able to transduce CAR-negative NIH 3T3 cells. The results also demonstrate that the PEG-g-PEI/Ad complexes are less toxic, less hemolytic, and more appropriately sized than PEI/Ad complexes. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

The Sleeping Beauty (SB) transposase system for somatic integration offers great potential for in vivo gene therapeutic applications and genome engineering. Until recently, however, efficacy of SB transposase as a gene transfer vector especially in large animals was lacking. Herein, we report about the newest viral vector development for delivery of the SB transposase system into large mammals. Over the past decade various hyperactive versions of SB transposase and advanced adenovirus vectors enabling efficient and safe delivery of transgenes in vivo were developed. Already several years ago it was demonstrated that adenovirus vectors can be used for delivery of the SB transposase system into murine liver. Our newest study showed for the first time that a hyperactive transposase system delivered by high-capacity adenoviral vectors can result in somatic integration of exogenous DNA in canine liver, facilitating stabilized transgene expression and phenotypic correction for up to three years in a canine model of human disease. In this review we discuss safety issues and further improvements of this adenovirus based hybrid vector system for somatic integration. In the future this approach paves new paths towards the possible cure of human genetic diseases and novel strategies for in vivo genome engineering in large mammals.

It has been previously shown that integrase-defective HIV-1-based gene vectors can serve, with moderate efficiency, as substrate for DNA transposition by a transiently expressed Sleeping Beauty (SB) transposase. Here, we describe the enhanced gene transfer properties of a HIV-1/SB hybrid vector that allows efficient DNA transposition, facilitated by the hyperactive SB100X transposase, from vector DNA intermediates in primary human cells. Potent transposase-dependent integration of genetic cargo carried by the hybrid HIV-1/SB vector (up to 160-fold above background) is reported in human cell lines as well as in primary human fibroblasts and keratinocytes. The efficiency of transgene integration in context of the newly developed hybrid vector is comparable with that of conventional lentiviral vectors (LVs). Integration profiles of integrating HIV-1-derived vectors and SB transposons mobilized from LVs are investigated by deep sequencing of a large number of integration sites. A significant bias of lentiviral integrations in genes is reported, confirming that biological properties of the viral integration machinery facilitate preferred insertion into actively transcribed genomic regions. In sharp contrast, lentiviral insertions catalyzed by the SB100X transposase are far more random with respect to genes. Based on these properties, HIV-1/SB vectors may become valuable tools for genetic engineering and therapeutic gene transfer.

Mesenchymal stem cells (MSCs) hold promise for cell therapy, and implantation of MSCs engineered with a baculovirus transiently expressing the growth factor can augment the bone repair. To prolong the baculovirus-mediated transgene expression, we developed hybrid baculovirus vectors exploiting the FLP/Frt-mediated recombination for circular episome formation. Transduction of human MSCs with the hybrid baculovirus vectors harboring the osteoinductive bmp2 gene substantially extended the BMP2 expression and improved the cellular osteogenic differentiation. To confirm the potential in the clinical setting, the present study evaluated the biosafety profile of human MSCs engineered by the hybrid vectors. We unraveled that transduction of MSCs with the hybrid baculovirus vectors slightly impeded the cell proliferation after transduction, probably due to the perturbation of cellular gene expression and induction of innate responses. Nonetheless, the hybrid baculovirus vectors did not compromise the cell viability and cellular differentiation. No transgene integration into the host chromosome and disruption of the karyotype of the MSCs were observed. Additionally, no upregulation of proto-oncogenes or downregulation of tumor suppressor genes occurred in the MSCs transduced with the hybrid baculovirus vectors. Neither did the transduced MSCs induce tumor formation in nude mice. This study not only supported the safety of MSCs for cell therapy but also implicated the potential of the human MSCs engineered by the hybrid baculovirus vectors for their applications in clinical scenarios necessitating sustained transgene expression.

Adenoviral vectors offer many advantages for cancer gene therapy, including high transduction efficiency, but safety concerns related to severe immunogenicity and other side effects have led to careful reconsideration of their use in human clinical trials. To overcome these issues, a strategy of generating hybrid vectors that combine viral and non-viral elements as more intelligent gene carriers has been employed. Here, we coated adenovirus (Ad) with an arginine-grafted bioreducible polymer (ABP) via electrostatic interaction. We examined the effect of ABP-coated Ad complex at various ABP molecules/Ad particle ratios. Enhanced transduction efficiency was observed in cells treated with cationic ABP polymer-coated Ad complex compared to naked Ad. We also examined the coating of Ad with ABP polymers at the optimal polymer ratio using dynamic light scattering and transmission electron microscopy. In both high and low coxsackie virus and adenovirus receptor (CAR)-expressing cells, ABP-coated Ad complex produced higher levels of transgene expression than cationic polymer 25 K PEI. Notably, high cytotoxicity was observed with 25 K PEI-coated Ad complex treatment, but not with ABP-coated Ad complex treatment. In addition, ABP-coated Ad complex was not significantly inhibited by serum, in contrast to naked Ad. Moreover, ABP-coated Ad complex significantly reduced the innate immune response relative to naked Ad, as assessed by interleukin-6 (IL-6) cytokine release from macrophage cells. Overall, our studies demonstrate that Ad complex formed with ABP cationic polymer may improve the efficiency of Ad and be a promising tool for cancer gene therapy.

We developed hybrid vectors employing high-capacity adenovirus as a first-stage carrier encoding all the components required for in situ production of a second-stage lentivirus, thereby achieving stable transgene expression in secondary target cells. Such vectors have never previously been tested in normal tissues, because of the scarcity of suitable in vivo systems permissive for second-stage lentivirus assembly. Here we employed a novel murine model in which endogenous liver tissue is extensively reconstituted with engrafted human hepatocytes, and successfully achieved stable transduction by the second-stage lentivirus produced in situ from first-stage adenovirus. This represents the first demonstration of the functionality of adenoviral-lentiviral hybrid vectors in a normal parenchymal organ in vivo.

Lentiviral vectors enter cells with high efficiency and deliver stable transduction through integration into host chromosomes, but their preference for integration within actively transcribing genes means that insertional mutagenesis following disruption of host proto-oncogenes is a recognized concern. We have addressed this problem by combining the efficient cell and nuclear entry properties of HIV-1–derived lentiviral vectors with the integration profile benefits of Sleeping Beauty (SB) transposase. Importantly, this integration enzyme does not exhibit a preference for integration within active genes. We generated integrase-deficient lentiviral vectors (IDLVs) to carry SB transposon and transposase expression cassettes. IDLVs were able to deliver transient transposase expression to target cells, and episomal lentiviral DNA was found to be a suitable substrate for integration via the SB pathway. The hybrid vector system allows genomic integration of a minimal promoter-transgene cassette flanked by short SB inverted repeats (IRs) but devoid of HIV-1 long terminal repeats (LTRs) or other virus-derived sequences. Importantly, integration site analysis revealed redirection toward a profile mimicking SB-plasmid integration and away from integration within transcriptionally active genes favored by integrase-proficient lentiviral vectors (ILVs).

We newly prepared lipopolyplexes containing N-lauroylsarcosine (LS) as a hybrid vector for pulmonary gene delivery via the systemic route. Lipopolyplexes were composed of polyethylenimine (PEI), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethlylammonium chloride (DOTMA), LS, and plasmid DNA (pDNA). The particle size of lipopolyplex of PEI, DOTMA, and pDNA (lipopolyplex 2P-2D) was not largely changed by the addition of LS, although the addition of LS decreased the high zeta potential. Lipopolyplexes containing LS with low zeta potential showed little aggregation with erythrocytes and low cytotoxicity. In HepG2 cells, lipopolyplexes containing LS showed high transgene efficiency comparable to lipopolyplex 2P-2D. After intravenous injection of the complexes into mice, lipopolyplexes containing LS showed high transgene efficiency, comparable to lipopolyplex 2P-2D. In particular, lipopolyplexes containing LS showed extremely high transgene efficiency in the lung. As a result of the analysis to identify optimum formulations, we discovered that LS contributed to the high transgene efficiency in the lung as 76.7% of the contribution index. These results suggest that lipopolyplexes containing LS are safe and useful gene delivery vectors with lung directivity., Journal of Controlled Release, 136(3), pp. 213-219

Autonomous parvoviruses possess an intrinsic oncotropism based on viral genetic elements controlling gene expression and genome replication. We constructed a hybrid vector consisting of the H1 parvovirus-derived expression cassette comprising the p4 promoter, the ns1 gene and the p38 promoter flanked by the adeno-associated viruses 2 (AAV2) inverted terminal repeats and packaged into AAV2 capsids. Gene transduction using this vector could be stimulated by coinfection with adenovirus, by irradiation or treatment with genotoxic agents, similar to standard AAV2 vectors. However, the latter were in most cases less efficient in gene transduction than the hybrid vector. With the new vector, tumor cell-selective increase in transgene expression was observed in pairs of transformed and non-transformed cells, leading to selective killing of the transformed cells after expression of a prodrug-converting enzyme. Preferential gene expression in tumor versus normal liver tissue was also observed in vivo in a syngeneic rat model. Comparative transduction of a panel of different tumor cell lines with the H1 and the H1/AAV hybrid vector showed a preference of each vector for distinct cell types, probably reflecting the dependence of the viral tropism on capsid determinants.

The trans-splicing (ts) and overlapping (ov) vectors expand the packaging capacity of adeno-associated virus (AAV). But their application depends on the inherent properties of the target gene. The ts vectors require an optimal gene-splitting site and the ov vectors require a highly recombinogenic domain. In order to overcome these limitations, we developed a hybrid dual (hd) vector system. In the hd vectors, we inserted a highly recombinogenic alkaline phosphatase (AP) sequence in the ts vectors to allow for transgene-independent reconstitution through homologous recombination of the AP sequences. We first tested the hybrid system with the LacZ gene. Both in the cell line (in vitro) and in the mouse muscle (in vivo), the hd vectors significantly outperformed the ts and ov vectors. In muscle, the transduction efficiency of the hybrid vectors reached 80% of that from the single intact vector. Southern blot confirmed AP sequence-mediated transgene reconstitution. In order to validate the hybrid system, we split the 6 kilobase (kb) mini-dystrophin gene at the exon 55/56 junction, a predicted poor site for the ts approach. In dystrophic mdx mouse muscle, the hd vectors yielded 5.6-fold higher transduction than the ts vectors did. Taken together, these data suggest that the hybrid system efficiently expresses large therapeutic genes that are poor candidates for the ts and ov approaches.

Recombinant retroviral vectors based upon simple gammaretroviruses, complex lentiviruses, or potentially nonpathogenic spumaviruses represent relatively well characterized tools that are widely used for stable gene transfer. Different members of the Retroviridae family have developed distinct and potentially useful features related to their life cycle. These natural differences can be exploited for specialized applications in gene therapy and could conceivably be combined to create future retroviral hybrid vectors, ideally incorporating the following features: an efficient, noncytopathic packaging system with low likelihood of recombination; serum resistance; an ability to pseudotype with cell-specific envelopes; high-fidelity reverse transcription before cell entry; unrestricted cytoplasmic transport and nuclear import; an insulated expression cassette; specific chromosomal targeting; and physiologic or regulated levels of transgene expression. We envisage that, compared to contemporary vectors, a hybrid vector combining these properties would have increased therapeutic efficacy and an enhanced biosafety profile. Many of the above goals will require the inclusion of nonretroviral components into vector particles or transgenes.

Most virus mediated transfection systems are efficient; however, their highly immunogenic properties do tend to cause clinical problems. HVJ-liposome vector is a hybrid vector consisting of liposome and inactivated sendai virus (hemagglutinating virus of Japan HVJ), which has been reported to be have a low immunogenicity, while it can also be repeatedly administered. To enhance the transfection efficiency, especially in the liver, we investigated the efficacy of total vascular exclusion (TVE) during the portal vein injection (PVI) of the vectors.beta-galactosidase and luciferase expression were used as reporter genes. Wistar rats were injected with HVJ-liposome through PVI without TVE (PVI group, n = 10) or PVI with TVE (PVI + TVE group, n = 10). The control rats were infused with equal volumes of saline through the portal vein (control group n = 12). The transfection efficiencies were assessed by beta-galactosidase staining and a luciferase assay. Biochemical and histological analyses were performed to evaluate the tissue toxicity after gene transfer.The reporter genes expression in the liver dramatically increased after PVI + TVE in comparison to after PVI alone (1.2 x 10(5)versus 1.5 x 10(4) RLU/mg protein, P0.05 according to a luciferase assay). Notably, the extrahepatic "leaky" transgene expression could be minimized by PVI + TVE, whereas the general condition remained unchanged according to both the biochemical parameters and histological findings.The present data indicate that PVI + TVE may thus facilitate the liver-specific gene delivery using the HVJ-liposome method and this modality might also be applicable to other gene transfer systems.

SummaryMerging tumor targeting and molecular-genetic imaging into an integrated platform is limited by lack of strategies to enable systemic yet ligand-directed delivery and imaging of specific transgenes. Many eukaryotic viruses serve for transgene delivery but require elimination of native tropism for mammalian cells; in contrast, prokaryotic viruses can be adapted to bind to mammalian receptors but are otherwise poor vehicles. Here we introduce a system containing cis-elements from adeno-associated virus (AAV) and single-stranded bacteriophage. Our AAV/phage (AAVP) prototype targets an integrin. We show that AAVP provides superior tumor transduction over phage and that incorporation of inverted terminal repeats is associated with improved fate of the delivered transgene. Moreover, we show that the temporal dynamics and spatial heterogeneity of gene expression mediated by targeted AAVP can be monitored by positron emission tomography. This new class of targeted hybrid viral particles will enable a wide range of applications in biology and medicine.

Adeno-associated virus (AAV) serotype 1 (AAV1) has been shown to be more effective than the well-studied AAV serotype 2 (AAV2) in muscle gene transfer. Replacement of amino acids 350 to 430 of AAV2 VP1 with the corresponding amino acids from VP1 of AAV1 resulted in a hybrid vector, termed AAV-221-IV, which behaved similarly to AAV1 in vitro and in vivo in muscle. Intramuscular injection of 1 × 1011 vector particles per mouse of hybrid vector carrying a human FIX transgene in CD4 knockout mice resulted in an average level of human FIX in the plasma of 450 ng/ml, 4- to 10-fold higher than in mice injected with an AAV2 vector carrying the same transgene, and 80% of the transgene levels in animals treated with the same dose of AAV1. DNA analysis of injected muscle showed a 10-fold higher copy number after gene delivery by the hybrid vector compared with AAV2. A comparison of total DNA versus DNA from intact virus particles suggests a higher stability of hybrid virus particles. These results suggest that changes in the AAV capsid have an effect on virus-cell receptor interaction, and also influence trafficking and processing of the virus particle in the cell. This “hybrid vector” retains the heparin-binding sites of AAV2 and, therefore, can be purified by passage through a heparin-Sepharose column with the same efficiency as AAV2. When tested in vivo, either in CD4 knockout mice or in a hemophilic mouse model, the heparin-purified hybrid vector showed >10-fold higher activity than similarly purified AAV2. This demonstrates the utility of this hybrid vector in the performance of large-scale heparin column purification to generate a vector with a high expression profile for muscle-directed gene delivery. Initiation of clinical studies with this hybrid vector may be facilitated because it differs from AAV2 by only nine amino acids.

Uncontrolled insertion of gene transfer vectors into the human genome is raising significant safety concerns for their clinical use. The wild-type adeno-associated virus (AAV) can insert its genome at a specific site in human chromosome 19 (AAVS1) through the activity of a specific replicase/integrase protein (Rep) binding both the AAVS1 and the viral inverted terminal repeats (ITRs). AAV-derived vectors, however, do not carry the rep gene and cannot maintain site-specific integration properties. We describe a novel hybrid vector carrying an integration cassette flanked by AAV ITRs and a tightly regulated, drug-inducible Rep expression cassette in the framework of a high-capacity, helper-dependent adenoviral (Ad) vector. Rep-dependent integration of ITR-flanked cassettes of intact size and function was obtained in human primary cells and cell lines in the absence of selection. The majority of integrations were site specific and occurred within a 1000-bp region of the AAVS1. Genome-wide sequencing of integration junctions indicates that nonspecific integrations occurred predominantly in intergenic regions. Site-specific integration was obtained also in vivo, in an AAVS1 transgenic mouse model: upon a single tail vein administration of a nontoxic dose of Ad/AAV vectors, AAVS1-specific integrations were detected and sequenced in DNA obtained from the liver of all animals in which Rep expression was induced by drug treatment. Nonrandom integration of double-stranded DNA can therefore be obtained ex vivo and in vivoby the use of hybrid Ad/AAV vectors, in the absence of toxicity and with efficiency compatible with gene therapy applications.

To confer adenovirus vectors (AdV), the feature of integration into the host cell genome hybrid vectors were characterized in vitro, which express vectors derived from the prototypic foamy virus (FV) in the backbone of a high-capacity AdV. FVs constitute a subfamily of retroviruses with a distinct replication pathway and no known pathogenicity. In the absence of envelope glycoprotein, the prototypic FV behaves like a retrotransposon, while it behaves like an exogenous retrovirus in its presence. Two principle types of vectors, which either allows the intracellular (HC-FAD-7) or, in addition, the extracellular (HC-FAD-2) pathway were constructed. In both chimeras the expression of the FV vector was controlled by the tetracycline-regulatable system. Hybrids were produced close to 10(10) infectious units/ml. By Southern blotting, the functionality of the hybrid vectors to generate host cell genomic integrants was shown. However, the efficiency of HC-FAD-7 to establish stable transgene expression was rather low, while around 70% of cells were stably transduced in secondary round following primary transduction with HC-FAD-2 at an MOI of 100. Given the benign characteristics of high-capacity adenovirus and FV vectors, hybrids based on HC-FAD-2 are probably suited for an in vivo application.

Classically, the 5′ and 3′ long terminal repeats (LTRs) are considered necessary but not sufficient for retroviral integration. Recently, we reported that inclusion of these and additional elements from Moloney murine leukemia virus (MoMLV) facilitated transgene integration, without retroviral integrase, when placed in an adenoviral context (AdLTR-luc vector) (Nat. Biotech. 18 (2000), 176; Biochem. Biophys. Res. Commun. 300 (2003), 115). To help understand this nonhomologous DNA recombination event, we constructed another vector, AdELP-luc, with 2.7 kb of MoMLV elements identically placed into an E1-deleted adenovirus type 5 backbone upstream of a luciferase cDNA reporter gene. Unlike AdLTR-luc, no MoMLV elements were placed downstream of the expression cassette. AdELP-luc readily infected epithelial cells in vitro. Southern hybridizations with DNA from cloned cells showed that disruption of the MoMLV sequences occurred. One cell clone, grown in vitro without any special selection medium for 9 months, exhibited stable vector integration and luciferase activity. Importantly, both Southern hybridization and FISH analyses showed that in addition to the MoMLV elements and expression cassette, substantial adenoviral sequence downstream of the luciferase cDNA was genomically integrated. These results suggest that the 2.7 kb of MoMLV sequence included in AdELP-luc have cis -acting functions and mediates an unusual integration event.

Activated telomerase is frequently detected in cancer cells and is able to maintain and stabilize the integrity of telomeres; it also contributes to unlimited divisions in cancer cells. Recently, a new generation of selective anticancer strategies is under development targeting the blockage of telomerase activity either at the protein level or telomerase RNA. Here, we report suppression of the malignant phenotype by the expression of the full-length antisense human telomerase RNA (hTR) delivered by a novel hybrid vector recombining adenovirus and adeno-associated virus (vAd-AAV). The hybrid vector vAd-AAV retained the unique traits from two parental viruses, such as high efficiency of gene transfer in mammalian cells and the ability to integrate into the genomic DNA of host cells. The stable expression of antisense hTR in MCF-7 cells significantly suppressed telomerase activity and progressively shortened telomere length for 30 population doublings (PD30). Expression of antisense hTR leads to a telomere-based growth arrest and the induction of spontaneous apoptosis. Antisense hTR decreased soft agar colony formation and reduced the cell proliferation, leading to exit from the cell cycle at G1 at PD15. The expression of antisense hTR also sensitized MCF-7 cells to apoptosis induced by sodium butyrate or serum starvation. Our study demonstrates that delivering antisense hTR by the hybrid Ad/AAV vector is an effective antineoplastic gene therapeutic strategy, which significantly suppresses the malignant phenotype and enhances apoptosis of human breast cancer cells.

The frequency with which the hybrid vector AdLTR-luc mediates genomic integration [Nat. Biotech. 18 (2000) 176–180] is unknown. To address this, we constructed AdLTR-red, using the AdLTR-luc backbone and the enhanced red fluorescence protein cDNA. Kinetic studies showed that AdLTR-red and a conventional adenoviral vector, AdCMV-red, entered and transduced epithelial cells comparably. AdLTR-red integration frequency in vitro, i.e., the percentage of red clones after 10–12 doubling times from limiting dilutions, was 8.0% (36/450; at 67 particles/cell). With AdCMV-red, 0/549 clones were integration-positive. Seven days after AdLTR-luc or AdCMV-luc (1011 particles) femoral vein administration to adult rats splenocytes were prepared, stimulated with concanavalin A, and examined by FISH. AdLTR-luc integration occurred in 5–11% of mitotic rat splenocytes, while no unequivocal integration was found with AdCMV-luc. These data provide the first quantitative evidence of the frequency for genomic integration with this hybrid vector.

Retroviral suicide gene vectors have successfully been used in clinical studies to improve the safety of adoptive immunotherapy with allogeneic T lymphocytes in the treatment of malignant and viral diseases. At the same time these studies have revealed several problems that are yet to be resolved including impaired T cell function due to long ex vivo culture. Here we present new retroviral vectors co-expressing truncated CD34, a gene transfer marker which ensures rapid enrichment of transduced cells using commercially available GMP-approved devices, and a splice-corrected variant of Herpes simplex virus thymidine kinase (scHSVtk) which confers high sensitivity to the prodrug ganciclovir. We show that a retroviral hybrid vector, MP71, based on the myeloproliferative sarcoma virus (MPSV) and the murine embryonic stem cell virus (MESV), encoding a tCD34/scHSVtk fusion protein mediates high expression of the 'sort-suicide' selection marker, thereby allowing for highly efficient purification and selective elimination of transduced cells.

TheabilitytotransferthedystrophingenestablytotheskeletalmuscleofDMDpatientsisamajorconfoundingissueinestablishinganeffectivegenetherapyforthisdisease.Toovercomethisproblem,wehaveexaminedtheabilityofmusclefibresfrommdx micetoactasin situ factoriesofretroviralvectorproduction.Tibialisanterior(TA)musclesfrom4-week-oldmdx micewereinjectedwithanadenoviralvectorexpressingLacZwithinaretroviralexpressioncassette(AdLZIN).Retroviralvectorproductionwasinducedbytheinclusionoftwoadditionaladenoviralvectorsexpressingretroviralgag–pol (AdGagPol)and10A1env genes(Ad10A1).Uponintroductionofinfectedmusclesintocellculture,coloniesofb-galactosidase-expressingmyotubesformedonlyincultureswherethemusclewasinjectedwithAdLZIN,AdGagPolandAd10A1,butnotfrommuscleinjectedwithAdLZINonly.Musclesfrommdx/nudemiceproducingretroviralvectordisplayeda4.6-foldincreaseinb-galactosidase-positivemyofibresafter1month,comparedwithcontralateralmuscleinthesameanimalinjectedwithAdLZINandAdGagPolonly.Byconstructingahybridadeno-retroviralvectorexpressingatruncated micro-dystrophin construct (AdlDyIN), we were able to partially correct the mdx dystrophicphenotype.AdlDyIN-mediatedexpressionofmicro-dystrophininmdx TAmusclerestoredtheformationofthedystrophin-associatedglycoproteincomplexandsignificantlyreducedthelevelofmuscledegenerationoveruninjectedcontrols.Bystimulating in situ production ofretroviralvectorexpressingmicro-dystrophin,weachieved92% 6%transductionofmyofibresintheTAmuscleby4weeks.Strikingly,by3monthspostinjection,micro-dystrophinwasstillexpressedtohighlevelsinnearlyallthemyofibresoftheTAmuscle.Bycomparison,therewasapronounceddropinthelevelsofmicro-dystrophinexpressedbymusclesinjectedwithAdlDyINonly.Finally, using a novel PCR approach, we detected reverse-transcribed, integrated proviral sequences inTAmusclegenomicDNAby4weekspostinjection,thelevelsofwhichwerefoundtoincreaseafter3months.INTRODUCTIONDuchennemusculardystrophy(DMD)isafatalX-chromosome-linked muscle wasting disease affecting human juvenile malesand caused by mutations in the 2.7Mb dystrophin gene. Itcurrently affects 1 in 3500 newborn males, one-third of thesecases being spontaneous (1). The dystrophin protein is a large(427kDa) membrane-associated protein that is thought to main-tain myofibre structural integrity by connecting the extracellularmatrix through the dystrophin-associated glycoprotein (DAG)

Herpes simplex virus type 1 (HSV-1)-based amplicon vectors have a large transgene capacity and can efficiently infect many different cell types. One disadvantage of HSV-1 vectors is their instability of transgene expression. By contrast, vectors based on adeno-associated virus (AAV) can either persist in an episomal form or integrate into the host cell genome, thereby supporting long-term gene expression. AAV expresses four rep genes, rep68 , - 78 , - 40 , and - 52 . Of those, rep68 or rep78 are sufficient to mediate site-specific integration of the AAV DNA into the host cell genome. The major disadvantage of AAV vectors is the small transgene capacity (∼4.6 kb). In this study, we constructed HSV/AAV hybrid vectors that contained, in addition to the standard HSV-1 amplicon elements, AAV rep68 , rep78 , both rep68 and - 78 , or all four rep genes and a reporter gene that was flanked by the AAV inverted terminal repeats (ITRs). Southern blots of Hirt DNA from cells transfected with the hybrid vectors and HSV-1 helper DNA demonstrated that both the AAV elements and the HSV-1 elements were functional in the context of the hybrid vector. All hybrid vectors could be packaged into HSV-1 virions, although those containing rep sequences had lower titers than vectors that did not. Site-specific integration at AAVS1 on human chromosome 19 was directly demonstrated by PCR and sequence analysis of ITR-AAVS1 junctions in hybrid vector-transduced 293 cells. Cell clones that stably expressed the transgene for at least 12 months could easily be isolated without chemical selection. In the majority of these clones, the transgene cassette was integrated at AAVS1, and no sequences outside the ITR cassette, rep in particular, were present as determined by PCR, ITR rescue/replication assays, and Southern analysis. Some of the clones contained random integrations of the transgene cassette alone or together with sequences outside the ITR cassette. These data indicate that the long-term transgene expression observed following transduction with HSV/AAV hybrid vectors is, at least in part, supported by chromosomal integration of the transgene cassette, both randomly and site specifically.

The helper-dependent adeno-associated virus type 2 (AAV-2) exhibits complex interactions with its helper adenovirus. Whereas AAV-2 is dependent on adenoviral functions for productive replication, it conversely inhibits adenoviral replication, both when its genome is present in trans after coinfection with both viruses and when it is present in cis , as in the production of recombinant adenovirus (rAd)/AAV-2 hybrid vectors. The notion that AAV-mediated inhibition of adenoviral replication is due predominantly to the expression of the AAV-2 Rep proteins was recently challenged by successful Rep78 expression in a rAd5 vector through recoding of the Rep open reading frame (ORF). We closely analyzed the relative contributions of AAV-2 nucleic acid elements and Rep protein expression to the inhibition of adenoviral replication in both of the above scenarios. When present in cis , a sequence element in the 3′ part of the rep gene, comprising only the AAV-2 p40 promoter and the AAV-2 intron sequence, which we termed the RIS-Ad, completely blocks adenoviral replication. p5/p19 promoter-driven Rep protein expression, on the other hand, only weakly inhibits rAd/AAV-2 vector propagation, and by inactivation of the RIS-Ad, it is feasible to generate first-generation rAd vectors expressing functional Rep proteins. The RIS-Ad plays no role in the inhibition of adenoviral replication in trans in a model closely mimicking AAV-2–Ad coinfection. In this case, expression of the Rep proteins is required, as well as the presence of an amplifiable inverted terminal repeat (ITR)-containing template. Thus, very different AAV-2 elements and mechanisms are involved in inhibition of adenoviral replication during rAd/AAV-2 vector propagation and after Ad-AAV coinfection. IMPORTANCE This is the first study to systematically compare the contributions of AAV-2 protein expression and AAV-2 nucleic acid elements to the inhibition of adenoviral replication in rAd/AAV-2 hybrid vector generation and in AAV-2–adenovirus coinfection. This study shows that the two inhibitory processes are very different with regard to AAV-2 functions and the mechanisms involved. Whereas inhibition of rAd/AAV-2 hybrid vector propagation mostly involves a 3′ nucleic acid element in the rep gene, inhibition of an adenoviral genome in trans requires the Rep proteins and the AAV ITRs. These findings have important implications both for a basic understanding of the AAV replication cycle and for generation of rAd/AAV-2 hybrid vectors expressing the nonstructural and structural proteins of AAV-2.

Adenovirus (Ad) is a potential vehicle for cancer gene therapy. However, cells that express low levels of the coxsackie and adenovirus receptor (CAR) demonstrate poor Ad infection efficiency. We developed a bile acid-conjugated poly(ethyleneimine) (DA3)-coated Ad complex (Ad/DA3) to enhance Ad transduction efficiency. The size distribution and zeta potential of Ad/DA3 increased to 324 ± 3.08 nm and 10.13 ± 0.21 mV, respectively, compared with those of naked Ad (108 ± 2.26 nm and -17.7 ± 1.5 mV). The transduction efficiency of Ad/DA3 increased in a DA3 polymer concentration-dependent manner. Enhanced gene transfer by Ad/DA3 was more evident in CAR-moderate and CAR-negative cancer cells. Competition assays with a CAR-specific antibody revealed that internalization of Ad/DA3 was not mediated primarily by CAR but involved clathrin-, caveolae-, and macropinocytosis-mediated endocytosis. Cancer cell death was significantly increased when oncolytic Ad and DA3 were complexed (RdB-KOX/DA3) compared to that of naked oncolytic Ad and was inversely proportional to CAR levels. Importantly, RdB-KOX/DA3 significantly enhanced apoptosis, reduced angiogenesis, reduced proliferation, and increased active viral replication in human tumor xenografts compared to that of naked Ad. These results demonstrate that a hybrid vector system can increase the efficacy of oncolytic Ad virotherapy, particularly in CAR-limited tumors.

Lentiviral vectors are attractive candidates for gene therapy because of their ability to integrate into nondividing cells. To date, conventional HIV-1-based vectors can be produced at higher titers, but concerns regarding their safety for human use exist because of the possibility of recombination leading to production of infectious virions with pathogenic potential. Development of lentivirus vectors based on nonhuman lentiviruses constitutes an active area of research. We described a novel HIV-SIV hybrid vector system in which an HIV-1-derived transfer vector is encapsidated by SIVmac1A11 core particles and pseudotyped with VSV glycoprotein G. In an effort to further develop this vector system, we modified the packaging plasmid by deletion of the SIV accessory genes. Specifically, versions of the packaging plasmid (SIVpack) lacking vif, vpr, vpx, and/or nef were constructed. Our results indicate that, as with HIV-1-based packaging plasmids, deletion of accessory genes has no significant effect on transduction in either dividing or nondividing cells. The SIV packaging plasmid was also modified with regard to the requirement for RRE and rev. Deletion of the RRE and rev from SIVpack led to dramatic loss of transduction ability. Introduction of the 5' LTR from the spleen necrosis virus to packaging plasmids lacking RRE/Rev was then sufficient to fully restore vector titer. A minimal SIV transfer vector was also developed, which does not require RRE/Rev and exhibits no reduction in transduction efficiency in two packaging systems. The SIV-based vector system described here recapitulates the biological properties of minimal HIV-1-derived systems and is expected to provide an added level of safety for human gene transfer. We suggest that the SIV-derived vector system will also be useful to deliver anti-HIV-1 gene therapy reagents that would inhibit an HIV-1-derived vector.

Many gene therapy indications would benefit from vectors capable of achieving efficient in vivo delivery and long-term transgene expression in either dividing or nondividing cells. Such vector systems are not yet available. To achieve both goals, we have used noncytotoxic E1- and E4-deleted adenoviral vectors as vehicles for delivering an Epstein-Barr virus-based self-replicating episome (replicon) via Cre/loxP site-specific recombination. Co-infection of human cells with a proreplicon-encoded and a Cre-expressing adenovirus resulted in efficient delivery and excision of a functional replicon in the absence of vector-induced cytotoxicity. In addition, replication and nuclear retention of the replicon in the cell progeny translated into a prolonged transgene expression in actively dividing cells, both in vitro and in vivo. Combining desired features from different viruses within a single hybrid vector system should expand the range of clinical indications currently amenable to gene transfer.

One of the greatest challenges to gene therapy is the targetting of gene delivery selectively to the sites of disease and regulation of transgene expression without adverse effects. Ultimately, the successful realization of these goals is dependent upon improvements in vector design. Over the years, viral vector design has progressed from various types of replication-defective viral mutants to replication-conditioned viruses and, more recently, to ‘gutted’ and hybrid vectors, which have, respectively, eliminated expression of non-relevant or toxic viral genes and incorporated desired elements of different viruses so as to increase the efficacy of gene delivery in vivo. This review will focus on the different viral and cellular elements which have been incorporated into virus vectors to: improve transduction efficiencies; alter the entry specificity of virions; control the fate of transgenes in the host cells; and regulate transgene expression. Copyright © 2000 John Wiley & Sons, Ltd.

To improve gene transfer to CNS neurons, critical elements of herpes simplex virus 1 (HSV-1) amplicons and recombinant adeno-associated virus (AAV) vectors were combined to construct a hybrid amplicon vector, and then packaged via a helper virus-free system. We tested the HSV/AAV hybrid amplicon vectors for transduction efficiency and stability of transgene expression (green fluorescent protein) in primary neuronal cultures from rat fetal ventral mesencephalon, in comparison with traditional HSV amplicon, AAV, or adenovirus (Ad) vectors at the same multiplicity of infection. The HSA/AAV hybrid vectors transduced the highest number of primary neurons in culture 2 days after infection. As compared with all other vectors tested, only hybrid vectors containing the AAV rep gene maintained the 2-day level of transgene expression over 12 days in culture. This rep-containing hybrid vector was then tested for efficiency and safety in the brain. One month after injection into adult rat striatum (1 x 10(6) transducing units injected), transgene expression was observed within the striatum (ranging from 564 to 8610 cells) and the substantia nigra (via retrograde transport, ranging from 130 to 809 neurons). The HSV/AAV hybrid amplicon vectors transduced predominantly neurons within the striatum, and showed transduction efficacy similar to and in many cases higher than that of HSV amplicon vectors. No immune response was observed in the HSA/AAV hybrid vector-injected brains, as determined by immune markers specific for helper T lymphocytes, cytotoxic T lymphocytes, and microglia. This HSV/AAV hybrid system shows high transduction efficiency and stability in culture. The effective and safe transgene delivery into the nigrostriatal system illustrates its potential for therapeutic application for neurologic disorders, such as Parkinson and Huntington disease.

EBV episomes are nuclear plasmids that are stably maintained through multiple cell divisions in primate and canine cells (J. L. Yates, N. Warren, and B. Sugden, Nature 313:812–815, 1985). In this report, we describe the construction and characterization of an E1-deleted recombinant adenovirus vector system that delivers an EBV episome to infected cells. This adenovirus-EBV hybrid vector system utilizes Cre-mediated, site-specific recombination to excise an EBV episome from a target recombinant adenovirus genome. We demonstrate that this vector system efficiently delivers the EBV episome and stably transforms a large fraction of infected canine D-17 cells. Using a colony-forming assay, we demonstrate stable transformation of 37% of cells that survive the infection. However, maximal transformation efficiency is achieved at doses of the E1-deleted recombinant adenoviruses that are toxic to the infected cells. Consequently, E1-deleted vector toxicity imposes a limitation on our current vector system.

Vectors based on herpes simplex virus type 1 (HSV-1) can efficiently transduce hepatocytes in the mouse liver, and vector genomes can persist for at least 2 months. However, 24 hr after gene transfer, the number of cells that express the transgene decreases rapidly and no transduced cells are detectable after 7 days. In this study, we examined the capability of a helper virus-free HSV/AAV hybrid amplicon vector to extend transgene expression in hepatocytes in vivo. HSV-1 amplicon or HSV/AAV hybrid amplicon vectors that express reporter genes from different transcriptional regulatory sequences were packaged into HSV-1 virions using a helper virus-free packaging system. To determine relative transduction efficiencies, vector stocks were titered on four different cell lines, including hamster kidney (BHK21) and human lung (Hs913T) fibroblasts, and mouse (G6Pase−/−) and human (NPLC) hepatocytes. After in vivo injection of vector stocks into mouse liver, tissue sections were examined for reporter gene expression and cellular inflammatory response. Blood samples were collected to measure serum transaminase levels as a biochemical index of liver toxicity. Expression of a reporter gene from liver-specific promoter sequences was consistently more effective in hepatic cells compared with fibroblasts, whereas the opposite was true when using an HSV-1 immediate-early promoter. Expression in hepatocytes in vivo was markedly longer from HSV/AAV hybrid vector compared with traditional HSV-1 amplicon vector: the number of transduced cells (∼2% of all hepatocytes) remained stable over 7 days after injection of HSV/AAV hybrid vector, whereas no transduced cells were detected 7 days after gene transfer with standard HSV-1 amplicon vector. The rapid decline in reporter gene expression from standard amplicons was not solely caused by a B or T lymphocyte-mediated immune response, as it also occurred in RAG2−/− mice. Hepatocyte toxicity and cellular inflammatory effects associated with HSV/AAV hybrid vector-mediated gene transfer were minimal, and readministration of vector stock proved equally effective in naive mice and in animals that received a first vector dose 4 weeks earlier. HSV/AAV hybrid amplicon vectors support gene expression in vivo for considerably longer than do traditional HSV-1 amplicon vectors. Moreover, expression from these vectors does not provoke an overt inflammatory or immune response, allowing efficacious expression following repeated in vivo dosing. These characteristics suggest that such vectors may hold future promise for hepatic gene replacement therapy.

The aim of this study was to investigate the cellular delivery mechanism of the hybrid vector comprising the recombinant adeno-associated virus (rAAV) and polyethylenimine (PEI). The rAAV vector, rAAV-rIns1-hInsM2-ΔEGFP, was fluorescently labeled with Cy3, a cyanine dye, and complexed with PEI. The interaction of the hybrid vector with the Huh7 hepatoma cells was monitored by confocal microscopy. Complexation of rAAV with PEI enhanced the transduction efficiency, which was decreased by pretreatment of the cells with sodium chlorate, an inhibitor of glycosaminoglycan sulfation, suggesting the roles of heparan sulfate proteoglycans (HSPG) in the uptake of the hybrid vector by the cells. Examination by flow cytometry and confocal microscopy demonstrated an enhanced interaction between the cells and the virus when complexed with PEI. Pretreatment with wortmannin or cytochalasin B significantly reduced the virus uptake by the cells, suggesting the involvement of phosphatidylinositol 3-kinase (PI3K) signaling and phagocytosis in the interaction between the cells and the hybrid vectors. Treatment of cells with the antioxidants, including l-ascorbic acid, δ-tocotrienol, or N-acetylcysteine (NAC), impaired the rAAV-PEI-mediated transduction. Results obtained in this study illustrated the involvement of PI3K/Akt signaling and the ROS production in gene delivery via the rAAV-PEI hybrid vector.

Recombinant retroviruses provide highly efficient gene delivery and the potential for sustained gene expression, but suffer from significant disadvantages including low titer, expensive production, poor stability and limited flexibility for modification of tropism. In contrast, polymer-based vectors are more robust and allow cell- and tissue-specific deliveries via conjugation of ligands, but are comparatively inefficient. The design of hybrid gene delivery agents comprising both virally derived and synthetic materials (nanobiovectors) represents a promising approach to development of safe and efficient gene therapy vectors. Non-infectious murine leukemia virus-like particles (M-VLPs) were electrostatically complexed with chitosan (χ) to replace the function of the viral envelope protein. At optimal fabrication conditions and compositions, ranging from 6 to 9 μg chitosan/109 M-VLPs at 10 × 109 M-VLPs/ml to 40 μg chitosan/109 M-VLPs at 2.5 × 109 M-VLPs/ml, χ/M-VLPs were ~ 300–350 nm in diameter and exhibited efficient transfection similar to amphotropic MLV vectors. In addition, these nanobiovectors were non-cytotoxic and provided sustained transgene expression for at least three weeks in vitro. This combination of biocompatible synthetic agents with inactive viral particles to form a highly efficient hybrid vector is a significant extension in the development of novel gene delivery platforms.

To improve the expression levels of transgenes in malignant hematopoietic cells, we developed a novel adenoviral-alphavirus hybrid vector Ad5/F11p-SFV-GFP that contains a Semliki Forest Virus (SFV) replicon and chimeric fibers of Ad5 and Ad11p. Ad5/F11p-SFV-GFP infected >95% of K562, U937 or Jurkat cells and 23.65% of HL-60 cells, and led to moderate Enhanced Green Fluorescent Protein (EGFP) transgene expression intensity. The infection efficiency of Ad5/F11p-SFV-GFP in primary human leukemia cells ranged from 9.34-89.63% (median, 28.58%) at a multiplicity of infection (MOI) of 100, compared with only 3.37-44.54% (median, 10.42%) in cells infected by Ad5/F11p-GFP. Importantly, Ad5/F11p-SFV-GFP led to a significantly higher transgene expression level in primary leukemia cells, as indicated by the relative fluorescence intensity, compared to cells infected with Ad5/F11p-GFP. The increased expression of EGFP in Ad5/F11p-SFV-GFP-infected cells was associated with the accumulation of abundant subgenomic mRNA. Additionally, infection of K562, U937 or Jurkat cells by Ad5/F11p-SFV-GFP was significantly inhibited by blocking CD46 receptor; however, other factors may affect the gene-transfer efficiency of Ad5/F11p-SFV-GFP in primary leukemia cells. In conclusion, we successfully developed a novel adenoviral-alphavirus hybrid vector with RNA replicon features, which represents a promising vector for gene modifications during the production of cell-based vaccines for leukemia patients.

Adenovirus and adeno-associated virus (AAV) are eukaryotic DNA viruses being developed as vectors for human gene therapy. The strengths of each system have been exploited in a novel vector that is based on an ade novirus–AAV hybrid virus incorporated into a plasmid-based molecular conjugate. Efficient rescue and replication of the recombinant AAV genome in this hybrid required transient expression of rep. This feature was incorporated into the transducing particle by conjugating a rep expression plasmid to the hybrid virus through a polylysine bridge. The resulting particle is an attractive vehicle for gene therapy because it is easily manufactured and capable of efficiently transducing cells with the end result being rescue and replication of the recombinant AAV genome. This particle is also useful in the production of recombinant AAV resulting in yields 10-fold greater than that achieved with transfection-based protocols.

In dividing cells, the two aims a gene therapeutic approach should accomplish are efficient nuclear delivery and retention of therapeutic DNA. For stable transgene expression, therapeutic DNA can either be maintained by somatic integration or episomal persistence of which the latter approach would diminish the risk of insertional mutagenesis. As most monosystems fail to fulfill both tasks with equal efficiency, hybrid-vector systems represent promising alternatives. Our hybrid-vector system synergizes high-capacity adenoviral vectors (HCAdV) for efficient delivery and the scaffold/matrix attachment region (S/MAR)-based pEPito plasmid replicon for episomal persistence. After proving that this plasmid replicon can be excised from adenovirus in vitro, colony forming assays were performed. We found an increased number of colonies of up to sevenfold in cells that received the functional plasmid replicon proving that the hybrid-vector system is functional. Transgene expression could be maintained for 6 weeks and the extrachromosomal plasmid replicon was rescued. To show efficacy in vivo, the adenoviral hybrid-vector system was injected into C57Bl/6 mice. We found that the plasmid replicon can be released from adenoviral DNA in murine liver resulting in long-term transgene expression. In conclusion, we demonstrate the efficacy of our novel HCAdV-pEPito hybrid-vector system in vitro and in vivo.Molecular Therapy-Nucleic Acids (2013) 2, e83; doi:10.1038/mtna.2013.11; published online 2 April 2013.

Oncolytic adenoviruses (Ads) have shown great promise in cancer gene therapy but their efficacy has been compromised by potent immunological, biochemical, and specific tumor-targeting limitations. To take full advantage of the innate cancer-specific killing potency of oncolytic Ads but also exploit the subtleties of the tumor microenvironment, we have generated a pH-sensitive and bio-reducible polymer (PPCBA)-coated oncolytic Ad. Ad-PPCBA complexes showed higher cellular uptake at pH 6.0 than pH 7.4 in both high and low coxsackie and adenovirus receptor-(CAR)-expressing cells, thereby demonstrating Ad-PPCBA's ability to target the low pH hypoxic tumor microenvironment and overcome CAR dependence for target cell uptake. Endocytic mechanism studies indicated that Ad-PPCBA internalization is mediated by macropinocytosis instead of the CAR-dependent endocytic pathway that internalizes naked Ad. VEGF-specific shRNA-expressing oncolytic Ad complexed with PPCBA (RdB/shVEGF-PPCBA) elicited much more potent suppression of U87 human brain cancer cell VEGF gene expression in vitro, and human breast cancer MCF7 cell/Matrigel plug vascularization in a mouse model, when cancer cells had been previously infected at pH 6.0 versus pH 7.4. Moreover, intratumorally and intravenously injected RdB/shVEGF-PPCBA nanocomplexes elicited significantly higher therapeutic efficacy than naked virus in U87-tumor mouse xenograft models, reducing IL-6, ALT, and AST serum levels. These data demonstrated PPCBA's biocompatibility and capability to shield the Ad surface to prevent innate immune response against Ad after both intratumoral and systemic administration. Taken together, these results demonstrate that smart, tumor-specific, oncolytic Ad-PPCBA complexes can be exploited to treat both primary and metastatic tumors.

Salivary glands are an attractive target for gene transfer. Salivary epithelial cells are considered to be highly differentiated and have low rates of cell division (~6 months), affording the opportunity to obtain relatively long-term transgene expression in the absence of genomic integration. Here, we report a novel modified hybrid adenoretroviral vector, which provides stable transgene expression in salivary epithelial cells in vivo for up to 6 months in the absence of genomic integration. This modified hybrid vector, Ad(ΔE1/3)LTR(2)EF1α-hEPO, encodes human erythropoietin (hEPO) and differs from a previously developed hybrid vector, AdLTR(2)EF1α-hEPO, by having more extensive E3 gene deletion. Following direct salivary gland gene transfer by retroductal cannulation, rats transduced with Ad(ΔE1/3)LTR(2)EF1α-hEPO had sustained, elevated serum hEPO levels and hematocrits for 6 months (length of experiment), as compared with ~2 months for animals administered the AdLTR(2)EF1α-hEPO vector. Immunohistochemistry demonstrated that this novel vector could transduce both acinar and ductal cells. Interestingly, the Ad(ΔE1/3)LTR(2)EF1α-hEPO vector evoked much weaker local (salivary gland) immune responses than seen after AdLTR(2)EF1α-hEPO vector delivery, which likely permits its significantly lengthened transgene expression in this tissue.

In order to enhance the nuclear import of the transgene, we prepared plasmid DNA/importin-β conjugates consisting of biotinylated poly(ethylenimine)s and recombinant streptavidin-fused importin-β. Hemagglutinating virus of Japan-envelope vector containing the PEI polyplex/importin-β conjugate showed high transfection efficiency not only in vitro but also in vivo. We showed that novel HVJ-E/importin-β-conjugated PEI polyplex hybrid vector could overcome plasma and nuclear membrane barriers to achieve effective transfection.

The pursuits of sustainable treatments for diseases and disorders that afflict the central nervous system (CNS) have proven challenging for the field of viral vector-based gene therapy. However, recent advances in viral vector technology coupled with efficient delivery methods have opened up new avenues that show promise at the preclinical testing stage. The development of the Herpes Simplex Virus/Sleeping Beauty (HSV/SB) hybrid vector represents such an advance for devising treatments targeting the CNS with its potential for stably integrating large transgenomic segments of DNA within the genomes of transduced cells. In utero administration of this hybrid vector into the embryonic mouse brain has revealed the capacity for widespread transgene dissemination due to the targeting of a neuronal precursor cell population. This unique feature has provided the means to stably express a transgene throughout the brain for prolonged periods, which is a prerequisite for the treatment of progressive CNS disorders. In this review we provide a comprehensive breakdown of the characteristics of the HSV/SB vector system and how it can be efficiently employed in the derivation of CNS-targeted gene therapeutic strategies.