Development of herpesvirus-based episomally maintained gene delivery vectors

Successful gene therapy aims to deliver and express therapeutic genes to cure or slow the progression of disease. However, a major obstacle in the application of gene therapy has been the development of the vectors used to deliver heterologous DNA to the cell or tissue of choice. A number of viral- and non-viral-based vector systems have undergone clinical trials with varying success. However, at present, no vector system possesses the full complement of properties that are generally believed necessary in an ideal gene delivery system. Therefore, alongside attempts to improve current gene delivery vectors, the identification and evaluation of new viral vectors is crucial for the long-term success of gene therapy. Herpesviruses are large DNA viruses which possess a number of advantages as gene delivery vectors. These relate to an ability to package large DNA insertions and establish lifelong latent infections in which the genomic material exists as a stable episome. This review aims to highlight the potential of herpesvirus vectors, in particular an alternative vector system based on herpesvirus saimiri (HVS). HVS is capable of infecting a range of human cell lines with high efficiencies, and the viral genome persists as high copy number, circular, non-integrated episomes which segregate to progeny following cell division. This allows the virus-based vector to stably transduce a dividing cell population and provide sustained transgene expression for an extended period of time both in vitro and in vivo. Moreover, the insertion of a bacterial artificial chromosome cassette into the HVS genome simplifies the incorporation of large amounts of heterologous DNA for gene delivery. These properties offer characteristics similar to an artificial chromosome combined with an efficient delivery system and merit its continual development as a possible gene delivery vector for the future.     

Stable marker gene transfer into human bone marrow stromal cells and their progenitors using novel herpesvirus saimiri-based vectors

We have evaluated the ability of new herpesvirus saimiri (HVS)-based vectors to deliver a marker gene green fluorescent protein (GFP) into human bone marrow (BM) stromal cells and their progenitors. Stromal cells expanded from adherent layers of long-term BM cultures (LTC) were susceptible to HVS-based infection in a dose-dependent manner, and the efficiency of 94.8 +/- 2.0% was achieved using single exposure with HVS/EGFP vector at multiplicity of infection (moi) of approximately 50. Colony-forming unit-fibroblast (CFU-F) assay established the ability of HVS-based vectors to infect progenitors for bone marrow stroma fibroblasts and transfer the marker gene over multiple cell divisions in the absence of selective pressure. HVS was not toxic for stromal cells and progenitors and no viral replication was detected upon growth of modified stroma. On the basis these data, we believe that HVS-based constructs can offer a new opportunity for selective gene delivery into bone marrow stromal cells and progenitors. The ability of HVS to infect nondividing cells can be considered advantageous in the development of both ex vivo and in vivo strategies.     

A novel system for mitigation of ectopic transgene expression induced by adenoviral vectors

Adenoviral (Ad) vectors are good candidates for gene therapy in view of their high in vivo gene delivery efficiency. However, greater control over the tissue distribution of transgene expression is required to avoid potentially deleterious effects in non-target organs. In this regard, the liver is particularly at risk due to the high natural tropism of Ad for this organ, where dose limiting toxicity has been seen due to toxic transgene expression. We hypothesized that the cre/loxP system could be utilized to reduce unintended transgene expression at this site. This concept was tested using an Ad vector (AdLCLLL) carrying a reporter gene cassette in which the promoter and luciferase gene were flanked by LoxP sequences. Co-administration of this vector with a second vector carrying the cre recombinase gene in vitro and in vivo resulted in specific down-regulation of transgene expression. This novel approach thus has the potential to improve the safety of gene therapy strategies that rely upon the delivery of genes which may be hepatotoxic.     

Herpesvirus saimiri-based gene delivery vectors maintain heterologous expression throughout mouse embryonic stem cell differentiation in vitro

In order to achieve a high efficiency of gene delivery into rare cell types like stem cells the use of viral vectors is presently without alternative. An ideal stem cell gene therapy vector would be able to infect primitive progenitor cells and sustain or activate gene expression in differentiated progeny. However, many viral vectors are inactivated when introduced in developing systems where cell differentiation occurs. To this end, we have developed a mouse in vitro model for testing herpesvirus saimiri (HVS)-based gene therapy vectors. We demonstrate here for the first time that HVS is able to infect totipotent mouse embryonic stem (ES) cells with high efficiency. We have transduced ES cells with a recombinant virus carrying the enhanced green fluorescent protein (EGFP) gene and the neomycin resistance gene (NeoR) driven by a CMV promoter and the SV40 promoter, respectively. ES cells maintain the viral episomal genome and can be terminally differentiated into mature haematopoietic cells. Moreover, heterologous gene expression is maintained throughout in vitro differentiation. Besides its obvious use in gene therapy, this unique expression system has wide ranging applications in studies aimed at understanding gene function and expression in cell differentiation and development.     

Immunity to adenovirus and adeno-associated viral vectors: implications for gene therapy

Viral vectors have provided effective methods for in vivo gene delivery for therapeutic purposes. The ability of viruses to infect a wide variety of cell types in vivo has been exploited for several applications, such as liver, lung, muscle, brain, eye and many others. Immune responses directed towards the viral capsids and the transgene products have severely affected the ability of these vectors to induce long-term gene expression. This paper reviews the influence of viral vectors on antigen-presenting cells (APC), which are central to the induction of innate as well as adaptive immune responses. In this respect, we have focused on adenovirus and adeno-associated viruses because of the polar responses these vector systems induce in vivo. While adenovirus vector can induce significant inflammatory responses, adeno-associated viral vectors are characterized by their inability to consistantly induce immune responses to the transgene product. Understanding the mechanism of infection, transduction and activation of APC by viral vectors will provide strategies to develop safe vectors and prevent immune responses in gene therapies.     

Liver-specific microRNA-122 target sequences incorporated in AAV vectors efficiently inhibits transgene expression in the liver

Vectors based on adeno-associated virus (AAV) are effective in gene delivery in vivo. Tissue-specific gene expression is often needed to minimize ectopic expression in unintended cells and undesirable consequences. Here, we investigated whether incorporation of target sequences of tissue-specific microRNA (miRNA) into AAV vectors could inhibit ectopic expression in tissues such as the liver and hematopoietic cells. First we inserted liver-specific miR-122 target sequences (miR-122T) into the 3'-untranslated region (UTR) of a number of AAV vectors. After intravenous delivery in mice, we found that five copies of the 20mer miR-122T reduced liver expression of luciferase by 50-fold and β-galactosidase (LacZ) by 70-fold. Five copies of miR-122T also reduced mRNA levels of a secretable protein (myostatin propeptide) from the AAV vector plasmid by 23-fold in the liver. However, gene expression in other tissues, including the heart was not inhibited. Similarly, we inserted four copies of miR-142-3pT or miR-142-5pT, both hematopoietic lineage-specific, into the 3'-UTR of the AAV-luciferase vector. We wished to see whether they could prolong transgene expression by inhibiting expression in antigen-presenting cells. However, in vivo luciferase gene expression in major tissues declined with time, regardless of the miR-142 target sequences used. Quantitative analysis of the vector DNA in various tissues revealed that the decline of transgene expression in vivo was mainly because of promoter shut-off other than loss of AAV-transduced cells by immune destruction. Moreover, transgene expression was not detected in circulating mononuclear cells after delivering AAV9 vector with or without miR142T. These results demonstrate that liver-specific miR-122 target sequence in AAV vectors was highly efficient in reducing liver expression, whereas hematopoietic miR-142 target sequences were ineffective in preventing decline of AAV vector gene expression in nonhematopoietic tissues resulted from promoter shut-off.     

Regulated and constitutive expression of anti-inflammatory cytokines by nontransforming herpesvirus saimiri vectors

Herpesviral saimiri-(HVS) mediated expression of bovine growth hormone was one of the first applications of an episomal viral vector for gene therapy. Meanwhile, the long-term persistence of HVS vectors has been confirmed in a broad spectrum of infectable target cells in vitro and in vivo. Regulated gene expression is useful for many applications of gene therapy. Therefore, we inserted the Mifepristone-antiprogestin-inducible expression system (GeneSwitchtrade mark) into HVS viral vectors to regulate the combined expression of anti-inflammatory cytokines, IL-10 and IL-1RA. Constitutive CMV-promoter/enhancer-driven and Mifepristone-inducible cytokine expression was compared in the viral context in transduced primary human fibroblasts and rheumatoid arthritis (RA) fibroblast-like cells (RASF). Long-term persistence of vector genomes was shown for both construct types. Constitutive expression was efficient and more rapid in onset than in the inducible system, in which the selective induction of interleukin expression along with low background levels was obtained by Mifepristone concentrations that were more than 1000-fold below those required for endogenous Progesterone antagonism. Furthermore, transgene expression corresponded to vector doses. Global patterns of cytokine secretion were not significantly changed due to viral transduction, indicating a rather inert behavior of the viral vector itself. In an attempt to emulate the inflammatory cytokine-enriched environment in rheumatoid arthritic joints, the function of the vectors could be demonstrated in vitro by the successful blockade of IL-1beta-stimulated matrix-metalloproteinase (MMP)-3 expression from RASF cells. Evaluation of this system in future studies, in suitable long-term SCID models of RA or in non-human primate models, will exploit the possible in vivo benefits of nontransforming HVS vectors in gene therapy.     

Rapid and highly efficient transduction by double-stranded adeno-associated virus vectors in vitro and in vivo

Adeno-associated virus (AAV) is a promising gene vector based on a single-stranded (ss) DNA virus. Its transgene expression requires the conversion of ssDNA to double-stranded (ds) genome, a slow process responsible for the delayed transduction and occasional inefficiency. By mutating the inverted terminal repeat, we have made novel AAV vectors that predominantly package the self-complementary dsDNA genome. The dsAAV consistently demonstrated superior and accelerated transduction in vitro and in vivo. Dramatic increases in transgene expression were observed in most of the cell lines examined, including B16 melanoma and 3LL lung cancer that are difficult to be transduced by the conventional ssAAV vectors. Similar increases were also observed in vivo in a variety of tissues including muscle and liver. The dsAAV transduced a vast majority of the hepatocytes for more than 6 months, while the ssAAV transduced only a small fraction. In addition to circumventing the requirement for DNA synthesis, the dsAAV exhibited higher in vivo DNA stability and more effective circularization than the ssAAV, suggesting potential molecular mechanisms for the faster, stronger and prolonged transgene expression.     

Spatial and temporal control of transgene expression through ultrasound-mediated induction of the heat shock protein 70B promoter in vivo

Adenoviral vectors have been constructed that express the transgenes luciferase (Adeno-HSP-Luc) or Fas ligand (Adeno-HSP-FasL) under the control of the heat shock protein 70B (hsp70B) promoter. Cultures infected with Adeno-HSP-Luc transiently expressed high levels of luciferase after heat shock. When cultures infected with Adeno-HSP-FasL were maintained at 37 degrees C, no transgene expression was observed, but when cultures were exposed to heat stress, transgene expression resulted in apoptotic cell death. In vivo, transgene expression was induced by ultrasound-mediated heating of adenovirus-infected tissue. In mice or rats injected with the Adeno-HSP-Luc construct, high levels of localized expression of luciferase activity were observed in regions subjected to ultrasound-mediated irradiation. Adeno-HSP-FasL was administered systemically to mice via the tail vein to evaluate safety. Animals receiving Adeno-HSP-FasL in the absence of ultrasound treatment did not display liver toxicity, whereas animals receiving ultrasound treatment to induce the expression of Fas ligand from the hsp70B promoter had significant increases in serum levels of liver enzymes. These data demonstrate that combining the inducible hsp70B promoter with ultrasound induction allows safe local expression of cytotoxic genes with possible therapeutic utility.     

AAV vectors transduce hepatocytes in vivo as efficiently in cirrhotic as in healthy rat livers

In liver cirrhosis, abnormal liver architecture impairs efficient transduction of hepatocytes with large viral vectors such as adenoviruses. Here we evaluated the ability of adeno-associated virus (AAV) vectors, small viral vectors, to transduce normal and cirrhotic rat livers. Using AAV serotype-1 (AAV1) encoding luciferase (AAV1Luc) we analyzed luciferase expression with a CCD camera. AAV1Luc was injected through the hepatic artery (intra-arterial (IA)), the portal vein (intra-portal (IP)), directly into the liver (intra-hepatic (IH)) or infused into the biliary tree (intra-biliar). We found that AAV1Luc allows long-term and constant luciferase expression in rat livers. Interestingly, IP administration leads to higher expression levels in healthy than in cirrhotic livers, whereas the opposite occurs when using IA injection. IH administration leads to similar transgene expression in cirrhotic and healthy rats, whereas intra-biliar infusion is the least effective route. After 70% partial hepatectomy, luciferase expression decreased in the regenerating liver, suggesting lack of efficient integration of AAV1 DNA into the host genome. AAV1Luc transduced mainly the liver but also the testes and spleen. Within the liver, transgene expression was found mainly in hepatocytes. Using a liver-specific promoter, transgene expression was detected in hepatocytes but not in other organs. Our results indicate that AAVs are convenient vectors for the treatment of liver cirrhosis.     

Transient foamy virus vector production by adenovirus vectors

The genome of the prototype foamy virus (PFV) has been introduced into an adenoviral/PFV hybrid vector and tested for stable in vitro gene transfer. Three different adenoviruses are used to encode: (i) the PFV structural genes gag and pol (Ad-GagPolDeltaPacI); (ii) the PFV structural gene env (Ad-Env); and (iii) the PFV vector genome (Ad-MD9) encoding the transgene (the enhanced green fluorescent protein (eGFP) gene). Following cotransduction by the three adenoviruses, the target cells become transient PFV vector-producing cells, resulting in the in situ release of recombinant PFV at a titre of up to 10(3) vector particles/ml, which can then infect surrounding cells, leading to stable integration of the expression cassette. Stable eGFP expression, observed for up to 60 days (11 passages) in cells transduced with all three adenoviral vectors, was shown by PCR to be the result of PFV integration. In contrast, cells transduced with only the adenovirus encoding the PFV vector genome showed a marked decrease in eGFP expression by passage 2 (16 days post-transduction) and did not contain integrated PFV vector. In short, this paper describes the production of a hybrid vector capable of high in vitro transduction and stable transgene expression using adenovirus and PFV vectors.     

High expression of transgenes mediated by hybrid retroviral vectors in hepatocytes: comparison of promoters from murine retroviruses in vitro and in vivo

To achieve high transgene expression in the liver, we have compared the reporter gene expression among various murine retroviral long terminal repeats (LTRs) or leader sequences in vitro. Transient reporter gene expression assays revealed the highest gene expression by the polycythemic strain of spleen focus-forming virus (SFFVp) LTR in differentiated hepatocellular carcinoma cell lines, HuH-7 and PLC/PRF/5. However, remarkable difference was not observed among LTRs in other types of human liver tumor cell lines. Essentially the same results were obtained by infecting these cells with a series of retroviral vectors. Repression of transgene expression was observed by the leader sequences from Moloney murine leukemia virus (MoMLV), but not from mouse embryonic stem cell virus (MESV). Strengths of the promoters were further compared in murine hepatocytes in vivo. Although the proportions of genomic integration were almost the same, higher gene expression was observed by the FMEV-type vector, which contained the SFFVp LTR and the MESV leader, in comparison with that by the MoMLV-based vector. Thus, FMEV-type vectors may represent a novel type of vectors for human gene therapy with hepatocytes.     

Constructing adenoviral vectors by using the circular form of the adenoviral genome cloned in a cosmid and the Cre-loxP recombination system.

Recombinant adenoviral vectors have been generated either by the in vivo homologous recombination method or by the in vitro direct ligation method. However, the efficiency of adenoviral vector construction by these methods is low, because of the large size of the recombinant vectors. To improve the ease of constructing adenoviral vectors, we used the circular form of adenoviral DNA, which can generate infectious viruses with an efficiency comparable to that of virion DNA, after transfection into 293 cells constitutively producing adenovirus E1 protein. We replaced the E1 region of the circular form of adenoviral DNA with a cosmid vector flanked by loxP sites, resulting in a 41-kb cosmid, designated pALC. An expression cassette that bicistronically expresses IL-5 and green fluorescent protein (GFP) was readily inserted between the loxP-flanked cosmid backbone and the adenoviral genome of pALC, using the cosmid vector cloning system. Transfection of the resulting cosmid into 293 cells did not produce any infectious adenoviruses because its size (46 kb) was larger than the packing capacity of the adenoviral particles. However, cotransfection of a Cre-expression plasmid with this cosmid into 293 cells efficiently excised the loxP-flanked cosmid vector backbone, and produced the adenoviral vector expressing IL-5 and GFP. To simplify our method further, we have produced a 293 cell line constitutively expressing Cre recombinase. Transfection of pALC cosmid alone into this cell line efficiently generated adenoviral vector. The adenoviral vector construction method presented here is simple and efficient and should further facilitate the application of recombinant adenoviral vectors for in vivo and in vitro gene transfer.     

Use of nonintegrating lentiviral vectors for gene therapy

Vectors based on lentiviruses have become potent tools for efficient gene transfer to multiple cell types both in vitro and in vivo. In part this is attributable to the stability of transduction afforded by integration into the target cell genome. However, evidence indicates that episomal forms of the vector can also be harnessed for effective gene expression. Nonintegrating vectors retain the high transduction efficiency and broad tropism of conventional lentiviruses but avoid the potential problems associated with the nonspecific integration of a transgene. In this respect they are particularly useful in postmitotic tissue because the vector genome is not diluted out through cell division. Here we discuss the various mutations that may be introduced into human immunodeficiency virus-based lentiviral vectors to achieve efficient transduction, and the mechanisms by which these vectors are effective. We also discuss their potential application to gene therapy and the treatment of genetic disease.     

Adenovirus vector-mediated gene transfer to regional lymph nodes

Regional lymph nodes (RLNs) possess important immune functions and represent a major pathway of metastasis for solid tumors. Given these facts, the ability to transfer exogenous genes to the RLNs with the goal of manipulating the local immunological milieu would be desirable. On the basis of the hypothesis that a significant proportion of adenovirus (Ad) gene transfer vectors traffic through the lymphatics, E1-E3- Ad vectors were injected into the hind footpad of C3H/He mice and the RLNs assessed for vector trafficking and transgene expression. A low dose (10(9) particles) of an Ad vector encoding the firefly luciferase gene (Ad-CMV.Luc) resulted in luciferase expression only in the injection site and RLNs, with no detectable systemic (liver, spleen, lung) expression. At a higher dose (10(11) particles), some expression could be detected systemically in addition to the RLNs, but at levels in liver 14-fold less than in the RLNs. Transgene expression in the RLNs was transient, peaking at 1 day, decreasing markedly by 7 days. At high doses (10(11) particles), interruption of draining lymphatics decreased the amount of systemic dissemination 22-fold, suggesting that a large proportion of the vector trafficks through the lymphatics before reaching the systemic circulation. Administration of a vector encoding the jellyfish green fluorescent protein gene (AdCMV.GFP, 10(11) particles) showed that transgene expression in the RLNs was primarily in the cortical area. After footpad injection of a fluorescent-labeled Ad vector (Cy3-AdCMV.Null), fluorescent virions were visualized in the draining lymph. Regional lymph collected from animals injected in the footpad with AdCMV.Luc (10(11) particles) contained functional vector. Augmentation of local immune function in the RLNs was achieved by footpad administration of an Ad vector encoding murine IL-12, resulting in high mIL-12 and IFN-gamma levels in the regional, but not distant, nodes. These data demonstrate that expression of exogenous genes in RLNs is easily accomplished with Ad vectors, Ad vector dissemination occurs primarily via the lymphatics after footpad administration in mice, and basic immune functions in the RLNs can be manipulated by Ad-mediated gene transfer in vivo.     

Effects of rate, volume, and dose of intratumoral infusion on virus dissemination in local gene delivery

Recent studies have shown that up to 90% of viral vectors could disseminate to normal organs following intratumoral infusion. The amount of dissemination might be dependent on the infusion conditions. Therefore, we investigated the effects of infusion rate, volume, and dose on transgene expression in liver and tumor tissues after intratumoral infusion of an adenoviral vector encoding luciferase. Luciferase expression was determined through bioluminescence intensity measurement. We observed that the luciferase expression in the liver was independent of the infusion rate but increased with the infusion dose, whereas the luciferase expression in the tumor was a bell-shaped function of the infusion rate. The latter observation was consistent with the distribution pattern of Evans blue-labeled albumin after its solution was infused into tumors at the same infusion rates. We also observed that the infusion volume could affect luciferase expression in the tumor but not in the liver. These observations implied that virus dissemination was determined mainly by the infusion dose, whereas the amount of transgene expression in the tumor depended on the distribution volume of viral vectors in the tumor as well as the infusion dose.     

Enhanced gene expression conferred by stepwise modification of a nonprimate lentiviral vector

The practical application of gene transfer as a treatment for genetic diseases such as cystic fibrosis or hemophilia has been hindered, in part, by low efficiencies of vector delivery and transgene expression. We demonstrated that a feline immunodeficiency virus (FIV)-based lentiviral vector pseudotyped with the envelope glycoprotein from the baculovirus Autographa californica (GP64) efficiently transduces and persistently expresses a reporter gene in respiratory epithelium in the absence of agents that disrupt cellular tight junction integrity. GP64-pseudotyped FIV also efficiently transduced murine hepatocytes after tail vein delivery. To improve the FIV-based vector, we tested the contribution of a series of modifications to luciferase expression in vitro and in vivo. These modifications included the addition of spleen necrosis virus U5 (SNV U5) and mutation of the major splice donor and gag start codon located in the packaging region of the FIV transgene plasmid. After vector modification, we observed significantly enhanced expression of luciferase in respiratory epithelia after nasal application and in the liver after tail vein delivery. In addition, we observed significantly enhanced human factor VIII production after tail vein delivery. These sequential modifications provide an improved FIV lentivirus platform for gene therapy applications and may be applied to other retroviral vectors.