Increased persistence of lung gene expression using plasmids containing the ubiquitin C or elongation factor 1alpha promoter.

For effective gene therapy of chronic disease, persistent transgene expression at therapeutic levels is required. Clinical studies of airway gene transfer in patients with cystic fibrosis (CF) have resulted in short-lived transgene expression. We used intra-nasal dosing of naked plasmid DNA to the murine lung as a model for investigating the duration of airway gene transfer from a series of reporter expression plasmids. Transgene expression was transient when mediated by the viral promoters CMV, RSV and SV40, falling to less than 10% of peak expression after 2 weeks, although the presence of the adenoviral E4ORF3 gene in cis, resulted in extended duration of reporter activity from the CMV promoter. Transient expression from these promoters was not due to loss of the vector as determined by quantitative TaqMan PCR analysis. However, use of the promoters from the human polybiquitin C (UbC) and the elongation factor 1alpha (EF1alpha) genes resulted in persistent gene expression in the mouse lung. The UbC promoter directed high-level reporter activity which was maintained for up to 8 weeks and was still detectable 6 months after a single administration. Such persistent airway transgene expression from a nonviral vector without the concomitant expression of a potential antigen has not been reported previously. Thus, despite the persistence of vector DNA in vivo, attenuation of promoter function may lead to silencing of transgene expression and careful selection of promoter sequences is recommended for in vivo gene transfer.     

Functional analysis of various promoters in lentiviral vectors at different stages of in vitro differentiation of mouse embryonic stem cells.

Given the therapeutic potential offered by embryonic stem (ES) cells, it is critical to optimize stable gene delivery and expression at different developmental stages of ES cell differentiation. Here, we systematically analyzed lentiviral vectors containing the following promoters: the human elongation factor 1alpha (EF1alpha) promoter, the human cytomegalovirus (CMV) immediate early region enhancer-promoter, the composite CAG promoter (consisting of the CMV immediate early enhancer and the chicken beta-actin promoter), the human phosphoglycerate kinase 1 (PGK) promoter, the murine stem cell virus (MSCV) long terminal repeat (LTR), or the gibbon ape leukemia virus (GALV) LTR. Our results show that the EF1alpha promoter directed robust transgene expression at every stage of mouse ES cell differentiation, whereas the CMV promoter drove transgene expression only during late stages. Similarly, the CAG and PGK promoters drove transgene expression at a significant level only during late stages. The MSCV LTR and the GALV LTR exhibited much lower promoter activities at all stages. Interestingly, mouse ES cells transduced with the EF1alpha promoter-containing lentiviral vector lost most of their transgene expression during in vitro differentiation to neural precursors and neuronal cells. Our results demonstrate that different cellular and viral promoters exhibit very distinct and dynamic properties not only in terms of promoter strength but also with respect to differentiation stage-specific activity.     

An internal polyadenylation signal substantially increases expression levels of lentivirus-delivered transgenes but has the potential to reduce viral titer in a promoter-dependent manner

In lentiviral gene delivery systems, transgene expression cassettes are commonly cloned without a polyadenylation signal to prevent disruption of full-length lentiviral genomes on mRNA maturation in producer cells. The lack of the polyadenylation signal, however, has the potential to reduce stability and translation efficiency of transgene mRNA. Therefore, we have assessed the effect of a strong internal polyadenylation [poly(A)] signal on both transgene expression levels in virus-infected cells and functional viral titer, in a series of eight self-inactivating lentiviruses expressing the mOrange transgene under the control of the constitutive cytomegalovirus (CMV), elongation factor 1alpha (EF1alpha), and beta-actin promoters or the highly tissue-specific prostate-specific antigen/probasin hybrid (PSA/Pb) promoter with or without a simian virus 40 (SV40) early polyadenylation signal downstream of the mOrange-coding sequence. We show that mOrange expression levels in virus-infected HEK-293, LNCaP, and primary prostate epithelial cells were increased 3- to 6.5-fold when an internal polyadenylation signal was present. When the CMV and EF1alpha promoters were used, functional viral titer decreased 8- to 9-fold in the presence of the polyadenylation signal, but titer was not affected when transgene expression was driven by the beta-actin promoter or tissue-specific PSA/Pb promoter. We therefore conclude that an internal polyadenylation signal in lentiviral vectors has a highly beneficial effect on transgene expression, but reduces viral titer in a promoter-dependent manner.     

Transactivator and structurally optimized inducible lentiviral vectors.

Lentiviral vectors offer well-recognized advantages as a gene delivery system both for the analysis of gene function and as a vehicle for gene therapy. In the present study optimized HIV-1-based vector systems that display efficient doxycycline (Dox)-dependent transgene expression in vitro and in vivo have been developed through the modification of factors that contribute to basal activity levels. Dissection of HIV-1 vectors harboring a tTA-dependent transgene expression cassette revealed several mechanisms that account for Dox-independent transgene expression, including those mediated by an internal CMV promoter, as well as a potential contribution from fusion proteins generated by translational readthrough. A precipitous reduction in basal activity levels was accomplished by separating the transactivator and the transgene cassettes into a binary vector system and by relocating the inducible promoter to the U3 region of the LTR. In addition, substituting the VP16 portion of tTA with the human p65 transactivating domain improved Dox-dependent transgene expression in a number of cell types. Optimizing HIV-1-based vectors culminated in a "toolbox" of vectors suitable for transgene delivery in vitro and in vivo, as conveyed by our ability to control the Dox-dependent differentiation of embryonic fibroblasts into muscle cells in vitro and transgene expression in rat brains.     

Tetracycline-regulated gene expression in replication-incompetent herpes simplex virus vectors

Although herpes simplex virus (HSV) vectors appear to have great potential as gene delivery vectors both in vitro and in vivo, the expression of foreign genes in such vectors cannot be easily regulated. Of the known eukaryotic regulatory systems, the tetracycline-inducible gene expression system is perhaps the most widely used because of its induction characteristics and because of the well-known pharmacological properties of tetracycline (Tet) and analogs such as doxycycline. Here, we describe the adaptation of the Tet-inducible system for use in replication-incompetent HSV vectors. HSV vectors were constructed that contained several types of Tet-inducible promoters for foreign gene expression. These promoters contained a tetracycline response element (TRE) linked to either a minimal cytomegalovirus (CMV) immediate-early promoter, a minimal HSV ICP0 promoter, or a truncated HSV ICP0 promoter containing one copy of the HSV TAATGARAT cis-acting immediate-early regulatory element (where R represents a prime base). All three promoter constructs were regulated appropriately by doxycycline, as shown by the expression of the marker gene lacZ in cell lines engineered to express Tet transactivators. The ICP0 promoter constructs expressed the highest and most sustained levels of lacZ, but the CMV promoter construct had the highest relative level of induction, suggesting their use in different applications. To extend the utility of Tet-regulated HSV vectors, vectors were constructed that coexpressed an inducible Tet transactivator in addition to the inducible lacZ marker gene. This modification resulted in tetracycline-inducible gene expression that was not restricted to specific cell lines, and this vector was capable of inducible expression in irreversibly differentiated NT2 cells (NT-neurons) for several days. Finally, HSV vectors were constructed that expressed modified Tet transactivators, resulting in improved induction properties and indicating the flexibility of the Tet-regulated system for regulation of foreign gene expression in HSV vector-infected cells.     

Development of a self-inactivating tet-on retroviral vector expressing bone morphogenetic protein 4 to achieve regulated bone formation.

The aims of this study were to explore the possibility of improving the design of self-inactivating (SI) retroviral vectors and to develop an SI vector that would allow optimal tet-on-regulated therapeutic gene expression. To minimize any interference between the viral promoter and the inducible promoter, we deleted different regulatory elements in the 3'LTR and examined their effects on transgene expression in transfected or transduced cells. In transfected cells, such deletions reduced the transgene expression. The insertion of a polyadenylation sequence could not completely compensate for this effect. We observed three patterns of transgene expression in cells transduced with these tet-on retroviral vectors: (1) high levels of both basal and inducible expression, (2) low levels of both basal and inducible expression, and (3) low levels of basal and high levels of inducible expression. After using the optimal vector to transduce muscle-derived stem cells, we were able to regulate the strong in vitro expression of transgenes-including enhanced green fluorescent protein and bone morphogenetic protein 4-via the addition or withdrawal of doxycycline (Dox). Implantation of the transduced cells and subsequent Dox-dependent induction of gene expression resulted in bone formation in vivo. Thus, we have developed an optimal SI retroviral vector that maintains a high titer, efficiently transduces muscle-derived stem cells, and enables both high levels of inducible gene expression in vitro and robust regulated bone formation in vivo.     

Efficient gene transfer to human peripheral blood monocyte-derived dendritic cells using human immunodeficiency virus type 1-based lentiviral vectors

Dendritic cells (DCs) are potent antigen-presenting cells and are capable of activating naive T cells. Gene transfer of tumor antigen and cytokine genes into DCs could be an important strategy for immunotherapeutic applications. Dendritic cells derived from peripheral blood monocytes do not divide and are therefore poor candidates for gene transfer by Moloney murine leukemia virus (Mo-MuLV)-based retroviral vectors. Lentiviral vectors are emerging as a powerful tool for gene delivery into dividing and nondividing cells. A three-plasmid expression system pseudotyped with the envelope from vesicular stomatitis virus (VSV-G) was used to generate lentiviral vector particles expressing enhanced green fluorescent protein (EGFP). Peripheral blood monocyte-derived DCs were cultured in the presence of GM-CSF and IL-4 and transduced with lentiviral or Mo-MuLV-based vectors expressing EGFP. FACS analysis of lentiviral vector-transduced DCs derived either from normal healthy volunteers or from melanoma patients demonstrated transduction efficiency ranging from 70 to 90% compared with 2-8% using Mo-MuLV-based vectors pseudotyped with VSV-G. Comparison of lentiviral vectors expressing EGFP driven by CMV or human PGK promoters showed similar levels of transgene expression. Lentiviral vector preparations produced in the absence of HIV accessory proteins transduced DCs at efficiencies equal to vectors produced with accessory proteins. Alu-HIV-1 LTR PCR demonstrated the genomic integration of the lentiviral vector in the transduced DCs. Transduced cells showed characteristic dendritic cell phenotype and strong allostimulatory capacity and maintained the ability to respond to activation signals such as CD40 ligand and lipopolysaccharide. These results provide evidence that lentiviral vectors are efficient tools for gene transfer and expression in monocyte-derived DCs that could be useful for immunotherapeutic applications.     

Characteristics of lentiviral vectors harboring the proximal promoter of the vav proto-oncogene: a weak and efficient promoter for gene therapy.

Recent published data have shown the efficacy of gene therapy treatments of certain monogenic diseases. Risks of insertional oncogenesis, however, indicate the necessity of developing new vectors with weaker or cell-restricted promoters to minimize the trans-activation activity of integrated proviruses. We have inserted the proximal promoter of the vav proto-oncogene into self-inactivating lentiviral vectors (vav-LVs) and investigated the expression pattern and therapeutic efficacy of these vectors. Compared with other LVs frequently used in gene therapy, vav-LVs mediated a weak, though homogeneous and stable, expression in in vitro-cultured cells. Transplantation experiments using transduced mouse bone marrow and human CD34(+) cells confirmed the stable activity of the promoter in vivo. To investigate whether the weak activity of this promoter was compatible with a therapeutic effect, a LV expressing the Fanconi anemia A (FANCA) gene was constructed (vav-FANCA LV). Although this vector induced a low expression of FANCA, compared to the expression induced by a LV harboring the spleen focus-forming virus (SFFV) promoter, the two vectors corrected the phenotype of cells from a patient with FA-A with the same efficacy. We propose that self-inactivating vectors harboring weak promoters, such as the vav promoter, will improve the safety of gene therapy and will be of particular interest for the treatment of diseases where a high expression of the transgene is not required.     

Evaluation of viral and mammalian promoters for use in gene delivery to salivary glands.

To optimize vectors for salivary gland gene transfer, we screened viral [cytomegalovirus (CMV; human immediate early), Rous sarcoma virus (RSV), simian virus 40, and Moloney murine leukemia virus long terminal repeat] and mammalian [elongation factor 1alpha (EF1alpha), cytokeratin 18 (K18), cytokeratin 19 (K19), kallikrein (Kall), and amylase (AMY), all human, and rat aquaporin-5 (rAQP5), and derivative elements] promoters driving luciferase activity in vitro and in vivo. In adenoviral vectors, the CMV promoter showed highest activity, with the EF1alpha and RSV promoters slightly less powerful, in rat submandibular glands (SMGs). The K18 2.5-kb, K19 3.0-kb, and rAQP5 0.4-kb and Kall promoters had intermediate activity, while the AMY promoter exhibited lowest activity. To localize transgene expression, enhanced green fluorescence protein was used. The CMV, RSV, EF1alpha, K18 2.5-kb, K19 3.0-kb, rAQP5 0.4-kb, and AMY promoters were not cell-type specific in SMGs; however, the Kall promoter was primarily active in ductal cells. These data will facilitate optimal expression cassette design for salivary gland gene transfer.     

Stability of lentiviral vector-mediated transgene expression in the brain in the presence of systemic antivector immune responses

Lentiviral vectors are promising tools for gene therapy in the CNS. It is therefore important to characterize their interactions with the immune system in the CNS. This work characterizes transgene expression and brain inflammation in the presence or absence of immune responses generated after systemic immunization with lentiviral vectors. We characterized transduction with SIN-LV vectors in the CNS. A dose-response curve using SIN-LV-GFP demonstrated detectable transgene expression in the striatum at a dose of 10(2), and maximum expression at 10(6), transducing units of lentiviral vector, with minimal increase in inflammatory markers between the lowest and highest dose of vector injected. Our studies demonstrate that injection of a lentiviral vector into the CNS did not cause a measurable inflammatory response. Systemic immunization after CNS injection, with the lentiviral vector expressing the same transgene as a vector injected into the CNS, caused a decrease in transgene expression in the CNS, concomitantly with an infiltration of inflammatory cells into the CNS parenchyma at the injection site. However, peripheral immunization with a lentiviral vector carrying a different transgene did not diminish transgene expression, or cause CNS inflammation. Systemic immunization preceding injection of lentiviral vectors into the CNS determined that preexisting antilentiviral immunity, regardless of the transgene, did not affect transgene expression. Furthermore, we showed that the transgene, but not the virion or vector components, is responsible for providing antigenic epitopes to the activated immune system, on systemic immunization with lentivirus. Low immunogenicity and prolonged transgene expression in the presence of preexisting lentiviral immunity are encouraging data for the future use of lentiviral vectors in CNS gene therapy. In summary, the lentiviral vectors tested induced undetectable activation of innate immune responses, and stimulation of adaptive immune responses against lentiviral vectors was effective in causing a decrease in transgene expression only if the immune response was directed against the transgene. A systemic immune response against vector components alone did not cause brain inflammation, possibly because vector-derived epitopes were not being presented in the CNS.     

Improved retinal transduction in vivo and photoreceptor-specific transgene expression using adenovirus vectors with modified penton base.

Adenovirus (Ad) vectors can be injected into human ocular tissues without producing adverse events and are therefore a promising means of gene transfer to the retina. However, when administered subretinally, Ad vectors primarily transduce the retinal pigment epithelium (RPE), whereas the majority of mutant gene products that cause photoreceptor (PR) degeneration are expressed exclusively in the PR cells. While it has been shown previously that pseudotyping of Ad can partially overcome the limited PR transduction by Ad5, we found that pseudotyping of Ad is not necessary for transduction of PR cells. We determined that, in the context of Ad, the cytomegalovirus (CMV) promoter is not significantly active in PRs. We compared expression levels from CMV and chicken beta actin (CBA) promoters in neural retina and found that CBA has a 173-fold greater potency than CMV. We also investigated the nature of the Ad-RPE interaction in murine retina and determined that the RGD domain in Ad penton plays a key role in RPE tropism. Deletion of the RGD domain coupled with use of the CBA promoter permitted transgene expression in neural retina approximately 667 times more efficiently than with Ad5 vectors. The use of these vectors in combination with a 4.7 kilobase (kb) rhodopsin promoter enabled transgene expression exclusively in PR cells in vivo.     

Variegation of retroviral vector gene expression in myeloid cells

We have comparatively evaluated the efficiency of a series of retroviral vectors transducing the gp91-phox gene, whose defects are responsible for impaired production of superoxide anion (O2-) by phagocytic cells and lead to the X-linked form of chronic granulomatous disease (X-CGD). These vectors included four constructs based on the MoMuLV backbone and expressing gp91-phox from the viral long terminal repeat (LTR) or from internal promoters, and one construct based on the myelotropic FMEV vector. Expression of the therapeutic gene from the MoMuLV LTR was unsatisfactory after transduction of the PLB985 X-CGD knockout cell line and of primary CD34+ hematopoietic progenitors from X-CGD patients. The presence of either constitutive or inducible internal promoters did not result in important improvements in the efficiency of O2- production and lowered the titers of the viral preparations. In contrast, sustained levels of superoxide generation were obtained upon transduction with the FMEV vector. To analyze the efficiency of transgene expression at the single cell level, over 150 cellular clones were generated from bulk cultures of PLB985 X-CGD cells transduced with this vector, each one representative of an individual transduction event. These clones revealed a markedly heterogeneous pattern of gp91-phox expression, ranging from complete silencing to full restoration of superoxide production. Within each clone, expression of the therapeutic gene correlated with the number of expressing cells rather than with the average levels of expression from each cell, indicating that at the single cell level, the proviral promoter is regulated by a binary, on/off mechanism. Moreover, both transduced bulk and clonal cell populations displayed a tendency to a progressive extinction of expression over time, with a mechanism involving LTR methylation. The design of novel retroviral vectors escaping silencing is highly desirable for efficient gene therapy.     

Lentivirus-mediated gene transfer and expression in established human tumor antigen-specific cytotoxic T cells and primary unstimulated T cells

In this report, we evaluated the efficiency of stable gene transfer into established CD8(+) human tumor antigen-specific cytotoxic T cell (CTL) lines and peripheral blood lymphocytes (PBL) by oncoretroviral and lentiviral vectors. In the oncoretroviral vector, the green fluorescent protein (GFP) reporter gene was regulated by the murine stem cell virus (MSCV) promoter. In three human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors, the GFP transgene was regulated by either a chimeric MSCV/HIV-1 promoter, or cellular promoters from human housekeeping genes PGK and EF1 alpha. We found that several lines of proliferating tumor-specific CTL were poorly (=2%) transduced by the oncoretroviral vector that transduced Jurkat T cell line efficiently (=80%). In contrast, three lentiviral vectors transduced 38-63% of these proliferating CTL. More interestingly, all lentiviral vectors packaged without the HIV-1 accessory proteins transduced human bulk PBL and purified CD4(+) and CD8(+) lymphocyte subsets without prior stimulation. Detailed analysis indicated that the lentiviral vectors containing the EF1 alpha or PGK ubiquitous promoter can transduce unstimulated PBL and achieve low-level transgene expression in the absence of any T-cell activation. However, T-cell activation subsequent to the transduction of unstimulated PBL is required for high-level transgene expression. Transduced PBL expressing transgene delivered by the lentiviral vectors still preserved resting and na?ve cell phenotypes. Taken together, prior T cell stimulation and HIV-1 accessory proteins are dispensable for lentivirus-mediated gene transfer into resting na?ve and memory T lymphocytes. These results will have significant implications for the study of T-cell biology and for the improvement of clinical gene therapies of acquired immune deficiency syndrome (AIDS) and cancer.