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PubMed2004

Hematopoietic stem cell gene therapy of murine protoporphyria by methylguanine-DNA-methyltransferase-mediated in vivo drug selection.

Erythropoietic protoporphyria (EPP) is an inherited defect of the ferrochelatase (FECH) gene characterized by the accumulation of toxic protoporphyrin in the liver and bone marrow resulting in severe skin photosensitivity. We previously described successful gene therapy of an animal model of the disease with erythroid-specific lentiviral vectors in the absence of preselection of corrected cells. However, the high-level of gene transfer obtained in mice is not translatable to large animal models and humans if there is no selective advantage for genetically modified hematopoietic stem cells (HSCs) in vivo. We used bicistronic SIN-lentiviral vectors coexpressing EGFP or FECH and the G156A-mutated O6-methylguanine-DNA-methyltransferase (MGMT) gene, which allowed efficient in vivo selection of transduced HSCs after O6-benzylguanine and BCNU treatment. We demonstrate for the first time that the correction and in vivo expansion of deficient transduced HSC population can be obtained by this dual gene therapy, resulting in a progressive increase of normal RBCs in EPP mice and a complete correction of skin photosensitivity. Finally, we developed a novel bipromoter SIN-lentiviral vector with a constitutive expression of MGMT gene to allow the selection of HSCs and with an erythroid-specific expression of the FECH therapeutic gene.

Animals↗
PubMed2002

Long-term bone marrow culture-derived stromal fibroblasts as a potential target for gene therapy in acute myelogenous leukemia.

As a part of our continuing efforts to develop gene therapy for acute myelogenous leukemia (AML), this study was undertaken to evaluate the possibility of using autologous bone marrow stromal fibroblasts (BMSFs) as a target cell population. Autologous BMSFs in AML were isolated from the stromal layers of long-term bone marrow culture (LTBMC) using immunomagnetic beads. BMSFs exhibited rapid proliferation even in the absence of growth factors. Cultures stimulated with bFGF produced significantly increased numbers of BMSFs than cultures without added growth factors. Using LNC/LacZ retroviral vector, the transduction efficiency of BMSFs was 13+/-4% at a 5 multiplicity of infection (MOI). LNC/interleukin-2 (IL-2)-transduced BMSFs produced between 1200 and 4800pg of IL-2/10(6) cells per 24h. Using adenoviral vector AdV/LacZ, the transduction efficiency was 84+/-10% at 100, and 92+/-8% at a MOI of 1000. Although the addition of basic fibroblast growth factor, epidermal growth factor, or platelet-derived growth factor did not affect the transduction efficiency, they increased the numbers of transduced cells significantly (P<0.01). AdV/IL-2-treated BMSFs produced high levels of IL-2 over the course of 7 days between 9820 and 22,700pg of IL-2/10(6) cells per 24h. Our finding that the genetically engineered autologous BMSFs of AML could be successfully established in vitro implies that BMSFs obtained from LTBMC might be considered as a target cell population for certain types of clinical gene therapy in AML.

Adenoviridae↗
PubMed2014

Genome-wide computational analysis reveals cardiomyocyte-specific transcriptional Cis-regulatory motifs that enable efficient cardiac gene therapy.

Gene therapy is a promising emerging therapeutic modality for the treatment of cardiovascular diseases and hereditary diseases that afflict the heart. Hence, there is a need to develop robust cardiac-specific expression modules that allow for stable expression of the gene of interest in cardiomyocytes. We therefore explored a new approach based on a genome-wide bioinformatics strategy that revealed novel cardiac-specific cis-acting regulatory modules (CS-CRMs). These transcriptional modules contained evolutionary-conserved clusters of putative transcription factor binding sites that correspond to a "molecular signature" associated with robust gene expression in the heart. We then validated these CS-CRMs in vivo using an adeno-associated viral vector serotype 9 that drives a reporter gene from a quintessential cardiac-specific &#x3b1;-myosin heavy chain promoter. Most de novo designed CS-CRMs resulted in a >10-fold increase in cardiac gene expression. The most robust CRMs enhanced cardiac-specific transcription 70- to 100-fold. Expression was sustained and restricted to cardiomyocytes. We then combined the most potent CS-CRM4 with a synthetic heart and muscle-specific promoter (SPc5-12) and obtained a significant 20-fold increase in cardiac gene expression compared to the cytomegalovirus promoter. This study underscores the potential of rational vector design to improve the robustness of cardiac gene therapy.

Animals↗
PubMed2000

Transfer of the feline erythropoietin gene to cats using a recombinant adeno-associated virus vector.

Chronic renal failure and the associated erythropoietin-responsive anemia afflicts over 2 million domestic cats in the United States, resulting in morbidity that can affect the owner-pet relationship. Although treatment of cats with recombinant human erythropoietin (Epo) protein can be effective, response to the drug often dissipates over time, probably due to the development of antibodies reactive with the human protein. As an alternate approach to the treatment of this disease, we have developed a recombinant adeno-associated virus vector containing the feline erythropoietin gene (rAAV/feEpo). This vector, when administered intramuscularly to normal healthy cats, caused a dose-related increase in hematocrit over a 7-week period after injection. Thus, the rAAV/feEpo vector holds promise as a simple, safe and effective therapy for the anemia of chronic renal failure in domestic cats.

Anemia↗
PubMed2004

Self-inactivating retroviral vectors with improved RNA processing.

Three RNA features have been identified that elevate retroviral transgene expression: an intron in the 5' untranslated region (5'UTR), the absence of aberrant translational start codons and the presence of the post-transcriptional regulatory element (PRE) of the woodchuck hepatitis virus in the 3'UTR. To include such elements into self-inactivating (SIN) vectors with potentially improved safety, we excised the strong retroviral promoter from the U3 region of the 3' long terminal repeat (LTR) and inserted it either downstream or upstream of the retroviral RNA packaging signal (Psi). The latter concept is new and allows the use of an intron in the 5'UTR, taking advantage of retroviral splice sites surrounding Psi. Three LTR and four SIN vectors were compared to address the impact of RNA elements on titer, splice regulation and transgene expression. Although titers of SIN vectors were about 20-fold lower than those of their LTR counterparts, inclusion of the PRE allowed production of more than 10(6) infectious units per ml without further vector optimizations. In comparison with state-of-the-art LTR vectors, the intron-containing SIN vectors showed greatly improved splicing. With regard to transgene expression, the intron-containing SIN vectors largely matched or even exceeded the LTR counterparts in all cell types investigated (embryonic carcinoma cells, fibroblasts, primary T cells and hematopoietic progenitor cells).

Animals↗
PubMed1995

Inhibition of intravascular thrombosis and vascular smooth muscle cell proliferation by gene therapy.

Intravascular thrombosis, smooth muscle cell proliferation and matrix production lead to arterial luminal narrowing and reduction of blood flow to various organs. Alteration of gene expression of the arterial wall cells to inhibit thrombosis and smooth muscle activation is emerging as a new and exciting therapeutic modality for cardiovascular pathology. We have used genetically modified endothelial cells to seed endovascular prostheses and tested cell adhesion to the prostheses both in vitro and in vivo. We also used two catheter-based systems to deliver genes directly to the arterial wall cells in vivo employing retroviral and adenoviral vectors. With efficient vectors for gene transfer and high level expression of proteins by the transduced cells, gene therapy will serve as a major therapy for post-angioplasty restenosis, unstable angina pectoris and vascular grafts stenosis.

Adenoviridae↗
PubMed2004

Transgene expression is increased by photochemically mediated transduction of polycation-complexed adenoviruses.

Poor efficiency of adenoviral gene transfer to target cells is a major limitation to adenoviral gene therapy. Inefficient gene transfer occurs in the absence of coxsackie- and adenovirus receptor (CAR) on the cell surface, and can be overcome by enhancing viral entry with cationic molecules. Recombinant adenovirus (Ad) noncovalently complexed with polycations imply a lack of transduction specificity. Therefore, we have investigated the potential of a novel light-specific treatment, named photochemical internalization (PCI), to enhance gene delivery of adenovirus serotype 5 (Ad5) complexed with the cationic agents poly-L-lysine (PLL) and SuperFect trade mark. Cell lines differing in their receptiveness to Ad5 were infected with amounts of virus transducing about 2% of the cells by conventional Ad infection. The combination of polycations and photochemical treatment enabled a substantial increase in reporter gene expression, resulting in up to 75% positive cells. The effect was most prominent in cell lines expressing moderate to low levels of CAR. Furthermore, we show that PCI enables proper gene delivery of fiberless Ad5 at viral concentrations and infection times where transduction of photochemically untreated cells was negligible, both in the absence and presence of PLL. Thus, we conclude that the photochemically induced transduction by adenoviral vectors complexed with polycations present an opportunity to obtain high cell-infectivity levels with low viral doses, also without the fiber-CAR interaction.

Adenocarcinoma↗
PubMed2005

Augmentation of the migratory ability of DC-based vaccine into regional lymph nodes by efficient CCR7 gene transduction.

Although dendritic cell (DC)-based immunotherapy is considered a promising approach for cancer treatment, a large quantity of DC vaccine is required for effective sensitization/activation of immune cells because of the poor migratory ability of administered DCs into regional lymphoid tissue. In this study, we created a DC vaccine sufficiently transduced with CC chemokine receptor-7 gene (CCR7/DCs) by applying RGD fiber-mutant adenovirus vector (AdRGD), and investigated its immunological characteristics and therapeutic efficacy. CCR7/DCs acquired strong chemotactic activity for CC chemokine ligand-21 (CCL21) and exhibited an immunophenotype similar to mature DCs but not immature DCs with regard to major histocompatibility complex/costimulatory molecule-expression levels and allogenic T cell proliferation-stimulating ability, while maintaining inherent endocytotic activity. Importantly, CCR7/DCs injected intradermally into mice could accumulate in draining lymph nodes about 5.5-fold more efficiently than control AdRGD-applied DCs. Reflecting these properties of CCR7/DCs, DC vaccine genetically engineered to simultaneously express endogenous antigen and CCR7 could elicit more effective antigen-specific immune response in vivo using a lower dosage than DC vaccine transduced with antigen alone. Therefore, the application of CCR7/DCs having positive migratory ability to lymphoid tissues may contribute to reduction of efforts and costs associated with DC vaccine preparation by considerably reducing the DC vaccine dosage needed to achieve effective treatment by DC-based immunotherapy.

Adenoviridae↗
PubMed1999

Reversal of diabetes in mice by implantation of human fibroblasts genetically engineered to release mature human insulin.

Autoimmune destruction of pancreatic beta cells in type I, insulin-dependent diabetes mellitus (IDDM) results in the loss of endogenous insulin secretion, which is incompletely replaced by exogenous insulin administration. The functional restoration provided by allogeneic beta-cell transplantation is limited by adverse effects of immunosuppression. To pursue an insulin replacement therapy based on autologous, engineered human non-beta cells, we generated a retroviral vector encoding a genetically modified human proinsulin, cleavable to insulin in non-beta cells, and a human nonfunctional cell surface marker. Here we report that this vector efficiently transduced primary human cells, inducing the synthesis of a modified proinsulin that was processed and released as mature insulin. This retrovirally derived insulin displayed in vitro biological activity, specifically binding to and phosphorylation of the insulin receptor, comparable to human insulin. In vivo, the transplantation of insulin-producing fibroblasts reverted hyperglycemia in a murine model of diabetes, whereas proinsulin-producing cells were ineffective. These results support the possibility of developing insulin production machinery in human non-beta cells for gene therapy of IDDM.

Animals↗
PubMed2000

Retroviral vector design studies toward hematopoietic stem cell gene therapy for mucopolysaccharidosis type I.

To optimize a gene transfer system for hematopoietic stem cell gene therapy of patients with mucopolysaccharidosis (MPS) type I, 10 retroviral vectors were constructed to express the human alpha-L-iduronidase (IDUA) cDNA. These vectors were designed to evaluate the potential effects of specific promoters, the addition of selectable markers, and the use of multiple promoters versus an internal ribosome entry site for expression of IDUA and selectable maker genes. The effect of vector design was investigated in primary patient fibroblasts (F(MPS)) or murine fibroblast cell lines; while overall comparison of transgene expression was determined in patients' peripheral blood lymphocytes (PBL(MPS)) and CD34+ progenitors (PBPC(MPS)). We observed that the human PGK promoter introduced the highest IDUA activity per 1% relative transgene frequency in F(MPS). Use of the same promoter to separately regulate both the therapeutic gene and a drug-resistance gene resulted in decreased expression of the unselected gene. Co-selection using bicistronic vectors not only increased the number of transductants, but also elevated transgene expression under selective pressure in transgene-positive progenitors. Bicistronic vector LP1CD overcame down-regulation and practically introduced the highest IDUA level in unselected PBL(MPS) and an intermediate level in PBPC(MPS). These studies provide a better understanding of factors contributing to efficient gene expression in hematopoietic cells.

Animals↗
PubMed2005

Retroviral vector backbone immunogenicity: identification of cytotoxic T-cell epitopes in retroviral vector-packaging sequences.

Retroviral vectors are the frequently applied gene delivery vehicles for clinical gene therapy, but specificity of the immunogenicity to the protein encoded by the inserted gene of interest is a problem which needs to be overcome. Here, we describe human cytotoxic T-lymphocyte (CTL) clones recognizing epitopes derived from the protein encoded by the retroviral vector backbone, which were established during the course of our attempts to generate CTLs against cytomegalovirus (CMV) or human papilloma virus (HPV) in vitro. In the case of healthy CMV-seronegative donors, CTL lines specific for retrovirally transduced cells were generated in four out of eight donors by stimulating CD8 T cells with CD40-activated B (CD40-B) cells retrovirally transduced with CMV-pp65. Two CTL clones derived from one of the CTL lines were found to recognize epitopes from gag in the context of HLA-B(*)4403 and -B(*)4601, respectively. Similarly, an HLA-B(*)3501-restricted CTL clone from a cervical cancer patient recognized an epitope located in the junctional regions of the gag and pol sequences. These results show that polypeptides encoded by components of the retroviral vector backbone are in fact immunogenic, generating CTLs in vitro in human cells. Thus, potential CTL responses to retroviral products should also be considered in clinical settings.

Animals↗
PubMed2006

Long-term persistence of gene expression from adeno-associated virus serotype 5 in the mouse airways.

Recombinant adeno-associated virus vectors based on serotype 2 (rAAV2) have been used to deliver transgenes to the airways in a variety of pre-clinical and clinical studies. Gene transfer in these studies has been severely restricted by the basolateral localization of rAAV2 receptors. Here, we studied vectors constructed from the AAV5 genome and capsid, which utilize N-linked sialic acid-containing receptors found on the apical surface of airway epithelial cells. We investigated gene transfer efficacy and duration of transgene expression following delivery of rAAV5/5 vectors to the mouse respiratory tract. Robust, dose-dependent transgene expression was observed in the epithelium lining the nose for at least 32 weeks, and for at least 52 weeks in the lung. Importantly, in the lung, transgene expression mediated by rAAV5/5 was 40-fold greater than by rAAV2/2 vectors. A distinct cellular preference for rAAV5/5-mediated transduction was observed, with transgene expression being predominantly restricted to sustentacular cells of the olfactory epithelium in the nose and alveolar type II cells in the lung. Administration of rAAV5/5 vectors to both the nose and lungs led to the rapid development of rAAV5/5-neutralizing antibodies, suggesting that repeated administration may be severely hampered by host immune responses.

Animals↗
PubMed2003

Gene therapy for ocular angiogenesis.

Ocular neovascularization is a central feature of diabetic retinopathy and age-related macular degeneration. These conditions are the major causes of blindness in the developed world. Current treatments are of limited efficacy and associated with significant adverse effects. Characterization of the molecular and cellular events involved in angiogenesis has led to the identification of a number of angiostatic molecules with potential therapeutic value. The systemic administration of small molecule angiostatic proteins risks significant systemic adverse effects and the effect of their intraocular injection is short-lived. Local gene transfer, however, offers the possibility of targeted, sustained and regulatable delivery of angiostatic proteins to the retina after a single procedure to introduce a vector to an intraocular site. The effect of intra-ocular delivery of recombinant viruses carrying genes encoding angiostatic proteins has been demonstrated in rodent models of ocular neovascularization. Recombinant adeno-associated virus-mediated local gene transfer of a vascular endothelial growth factor inhibitor controls both retinal and choroidal neovascularization. The clinical application of this approach may require the means to regulate gene expression in order to minimize the potential for adverse effects. Regulation of transgene expression by means of a hypoxia-responsive promoter offers an attractive strategy for the targeted and regulated delivery of angiostatic proteins to the retina in the management of ischaemia-induced ocular neovascularization. Preclinical studies of gene transfer in a large animal model following subretinal delivery of a recombinant adeno-associated virus vector have demonstrated efficient sustained reporter gene expression in cells of the outer retina. Recent progress has enabled the planning of clinical trials of gene therapy for ocular neovascular disorders.

Aged↗
PubMed2000

HSV vector cytotoxicity is inversely correlated with effective TK/GCV suicide gene therapy of rat gliosarcoma.

Herpes simplex virus (HSV)-mediated delivery of the HSV thymidine kinase (tk) gene to tumor cells in combination with ganciclovir (GCV) administration may provide an effective suicide gene therapy for destruction of malignant glioblastomas. However, because HSV is a highly cytotoxic agent, gene expression from the virus is short-lived which may limit the effectiveness of HSVtk/GCV therapy. Using different replication-defective HSVtk gene vectors, we compared HSV vector backgrounds for their cytotoxic activity on infection of 9L gliosarcoma cells in culture and brain tumors in rats and evaluated the impact of vector toxicity on the effectiveness of tk/GCV-mediated suicide gene therapy. As reported previously for other cell lines, a vector deleted for both copies of the immediate-early (IE) gene ICP4 (SOZ.1) was highly toxic for 9L cells in culture while a vector deleted in addition for the ICP22 and ICP27 IE genes (T.1) reduced or arrested 9L cell proliferation with more limited cell killing. Nevertheless, both vectors supported widespread killing of uninfected cells in the presence of GCV following low multiplicity infections, indicating that vector cytotoxicity did not preempt the production of vector-encoded TK enzyme necessary for the killing of uninfected cells by the HSV-tk/GCV bystander effect. Although an SOZ.1-related vector (SHZ.2) caused tumor cell necrosis in vivo, injection of SHZ.2 at multiple coordinates thoughout the tumor followed by GCV administration failed to prolong markedly the survival of tumor-bearing rats. In contrast, a single injection of T.1 produced a life-extending response to GCV. These results indicate that vector cytotoxicity can limit the efficacy of HSV-tk/GCV treatment in vivo, which may be due to premature termination of tk gene expression with attendant abortion of the bystander effect.

Animals↗
PubMed2006

Herpes simplex virus RNAi and neprilysin gene transfer vectors reduce accumulation of Alzheimer's disease-related amyloid-beta peptide in vivo.

Accumulation of insoluble aggregates of amyloid-beta peptide (Abeta), a cleavage product of amyloid precursor protein (APP), is thought to be central to the pathogenesis of Alzheimer's disease (AD). Consequently, downregulation of APP, or enhanced clearance of Abeta, represent possible therapeutic strategies for AD. We generated replication-defective herpes simplex virus (HSV) vectors that inhibit Abeta accumulation, both in vitro and in vivo. In cell culture, HSV vectors expressing either (i) short hairpin RNA directed to the APP transcript (HSV-APP/shRNA), or (ii) neprilysin, an endopeptidase that degrades Abeta (HSV-neprilysin), substantially inhibited accumulation of Abeta. To determine whether these vectors showed similar activity in vivo, we developed a novel mouse model, in which overexpression of a mutant form of APP in the hippocampus, using a lentiviral vector (LV-APP(Sw)), resulted in rapid Abeta accumulation. Co-inoculation of LV-APP(Sw) with each of the HSV vectors showed that either HSV-APP/shRNA or HSV-neprilysin inhibited Abeta accumulation in this model, whereas an HSV control vector did not. These studies demonstrate the utility of HSV vectors for reducing Abeta accumulation in the brain, thus providing useful tools to clarify the role of Abeta in AD that may facilitate the development of novel therapies for this important disease.

Alzheimer Disease↗
PubMed2004

Herpes simplex virus type 1 amplicons and their hybrid virus partners, EBV, AAV, and retrovirus.

HSV-1 amplicons can accommodate foreign DNA of any size up to 150 kbp. Genomic sequences as well as cDNA, large transcriptional regulatory sequences for cell type-specific expression, or multiple transgenes can be inserted in a modular fashion. HSV-1 amplicon vectors deliver DNA efficiently into the cell nucleus as an extrachromosomal, non-replicating circular concatenate, which is rapidly diluted, at least in dividing cells. Consequently, transgene expression is lost within days to weeks in dividing cells, but may be retained for months in non-dividing cells. In contrast, vectors based on Epstein-Barr virus, adeno-associated virus, or retroviruses can mediate long-term transgene expression, as vector DNA is retained by episomal replication or chromosomal integration. Hybrid amplicons use genetic elements from HSV-1 that allow replication and packaging of the vector DNA into HSV-1 virions, thereby conserving the large transgene capacity of HSV-1, and genetic elements from other viruses that confer genetic stability to the vector DNA within transduced cells. Additional strategies to sustain genetic material in infected cells include the incorporation of recombinases from different bacteriophages or transposable elements of the Tc1/mariner family in the amplicon vector. Moreover, modification of the HSV-1 virion itself offers a myriad of possibilities to improve gene delivery by targeting specific cell populations or transporting foreign proteins, such as Cre recombinase or the adeno-associated virus Rep protein, which can control the fate and expression of the therapeutic transgene.

Animals↗
PubMed2002

Oncolytic herpes simplex virus vectors for cancer virotherapy.

Oncolytic herpes simplex virus type 1 (HSV-1) vectors are emerging as an effective and powerful therapeutic approach for cancer. Replication-competent HSV-1 vectors with mutations in genes that affect viral replication, neuropathogenicity, and immune evasiveness have been developed and tested for their safety and efficacy in a variety of mouse models. Evidence to-date following administration into the brain attests to their safety, an important observation in light of the neuropathogenicity of the virus. Phase I clinical traits of three vectors, G207, 1716, and NV1020, are either ongoing or completed, with no adverse events attributed to the virus. These and other HSV-1 vectors are effective against a myriad of solid tumors in mice, including glioma, melanoma, breast, prostate, colon, ovarian, and pancreatic cancer. Enhancement of activity was observed when HSV-1 vectors were used in combination with traditional therapies such as radiotherapy and chemotherapy, providing an attractive strategy to pursue in the clinic. Oncolytic HSV-1 vectors expressing "suicide" genes (thymidine kinase, cytosine deaminase, rat cytochrome P450) or immunostimulatory genes (IL-12, GM-CSF, etc.) have been constructed to maximize tumor destruction through multimodal therapeutic mechanisms. Further advances in virus delivery and tumor specificity should improve the likelihood for successful translation to the clinic.

Animals↗
PubMed2005

Gene therapy for human small-cell lung carcinoma by inactivation of Skp-2 with virally mediated RNA interference.

Increase of Skp-2, which is involved in the degradation of cell cycle regulators including p27Kip1, p21 and c-myc, is one of the important mechanisms for dysregulation of cell cycles in various cancers. We applied RNA interference (RNAi) for Skp-2 by using HIV-lentiviral or adenoviral vectors for a human small-cell lung carcinoma cell line with increased Skp-2 to evaluate RNAi strategy for cancer gene therapy. HIV-lentivirus-mediated RNAi for Skp-2 resulted in efficient inhibition of the in vitro cell growth of cancer cells with increased Skp-2 through the increase of p27Kip1 and p21, but no significant effect on the growth of cells without high Skp-2 expression. Furthermore, intratumoral administration of adenovirus siRNA vector for Skp-2 efficiently inhibited growth of established subcutaneous tumor on NOD/SCID mice. These results indicate that the Skp-2 RNAi may be a useful strategy for gene therapy of cancers with high Skp-2 expression.

Adenoviridae↗