Human Gene Therapy

Hemophilia A and hemophilia B are both X chromosome-linked recessive bleeding disorders that affect about 1 in 5000 males and result from a deficiency in the coagulation factors VIII and IX, respectively. Severely affected individuals require frequent administration of factor VIII or factor IX preparations derived from human plasma, and more recently, from recombinant DNA technology. Although these preparations have greatly reduced the contamination with blood-borne pathogens, there still exist significant limitations with protein replacement therapy. As we elucidate more about the expression, structure, and function of these coagulation factors new avenues will open for the development of novel genetically improved coagulation factors. Several aspects of the hemophilias A and B make these diseases attractive candidates for gene therapy. These advantages include the following: (1) these proteins are readily delivered into the circulation from a variety of different cell types; (2) low levels of expression would significantly improve the management of bleeding episodes in these patients; (3) it is unlikely that regulated expression of these proteins will be required; and (4) there are excellent animal models for these diseases. Although progress with gene transfer of factor IX has proceeded at a greater rate than factor VIII, success with both molecules has demonstrated partial persistent correction in mouse and canine models of hemophilia A and B. The information gained from these animal studies has provided new insights into gene therapeutic approaches for genetic diseases. In addition, several gene therapy clinical studies for the treatment of hemophilia A and B were initiated in 1999.

Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) is a highly aggressive malignancy caused by the bcr-abl translocation oncogene. To explore alternative treatments for Ph+ ALL we tested gene-modified cell vaccines in the BALB/c-derived BM185 leukemia model. We compared the efficacy of BM185 cell vaccine expressing CD80 alone or in combination with IL-2 or GM-CSF. Mice injected with viable BM185 leukemia cells modified to express CD80 and GM-CSF (BM185/CD80+GM-CSF) showed the highest leukemia rejection rates. Cell vaccines consisting of irradiated BM185/CD80+GM-CSF cells administered subcutaneously stimulated a potent cytotoxic T lymphocyte (CTL) response against parental BM185. Histological examination of the vaccination site showed a large concentration of immune cells. Administration of the BM185/CD80+GM-CSF cell vaccine before intravenous challenge with parental cells caused strong inhibition of leukemia development. Vaccination after subcutaneous challenge with BM185 cells caused efficient elimination of leukemia promoting 40-60% long-term survival rates. The immunization efficacy of the BM185/CD80+GM-CSF cell vaccine was directly correlated with the percentage of cells expressing the transgenes. In all, this preclinical study shows that leukemia cell vaccines coexpressing CD80 and GM-CSF can potentially be explored for immunotherapy in Ph+ ALL patients.

While replication-defective retroviral vectors provide excellent vehicles for the long-term expression of therapeutic genes, they also harbor the potential to induce undesired genetic changes by random insertions into the host genome. The rate of insertional mutagenesis for retroviral vectors has been determined in several different assay systems; however, the rate at which such events induce cellular transformation has not been directly determined. Such measurements are critical to determining the actual risk of carcinogenesis resulting from retroviral gene therapy. In this study, the ability of a replication-defective retroviral vector, G1nBgSvNa, to induce cellular transformation in the BALB/c-3T3 in vitro transformation assay was assessed. The transformation frequency observed in vector-transduced BALB/c-3T3 cells, which contained one to six copies of integrated provirus, was not significantly different from that of untreated control cells. The finding that G1nBgSvNa was nontransforming in this assay indicates that the rate of transformation induced by retroviral insertions is less than the spontaneous rate of cellular transformation by BALB/c-3T3 cells, or less than 1.1 X 10-5. These results are the first to define an upper limit for the rate of transformation induced by retroviral vectors.

Adeno-associated virus (AAV) vectors have been shown to preferentially transduce hepatocytes after systemic administration in adult mice and to provide long-term expression of introduced genes. One application of this technology would be for the production of granulocyte colony-stimulating factor (G-CSF), which increases mature neutrophil numbers in humans and in animals, and has therapeutic effects in disorders featuring chronic neutropenia, including cyclic, severe congenital, and idiopathic neutropenia, and glycogen storage disease type Ib. We have treated mice by tail vein injection of AAV vectors encoding human G-CSF, and have detected high G-CSF levels and marked elevation of neutrophil counts for at least 5 months. A therapeutically relevant amount of G-CSF production was obtained when the liver-specific mouse albumin promoter-enhancer was used to drive G-CSF expression. In mice receiving higher amounts of vector, plasma levels of human G-CSF gradually increased over 3 weeks to high concentrations, whereas for lower amounts human G-CSF remained at initial, low levels. The previously observed effect of gamma irradiation, to increase AAV transduction rates, was diminished when large amounts of vector were used. Absolute neutrophil counts increased 10- to 50-fold for the period of observation to levels that would be therapeutic in the treatment of cyclic neutropenia. In conclusion, gene therapy with AAV vectors synthesizing G-CSF shows promise for the treatment of disorders featuring neutropenia.

We have shown that Flk2/Flt3 ligand (Flt3L)-transduced tumor vaccine induces transferable T cell protection against a murine breast cancer cell line, but a direct comparison with the potent effector GM-CSF, the activity against preestablished tumors, and the mechanism of antitumor response in this breast cancer model are not known. We compared vaccination with C3L5 cells expressing Flt3L (C3Lt-Flt3L) and GM-CSF(C3L5-GMCSF) by injecting 1 X 10 4 cells subcutaneously into the chest wall and then, after 4 weeks, challenging the contralateral chest of tumor-free mice with parental C3L5 cells. C3L5-Flt3L and C3L5-GMCSF had reduced in vivo growth rates (25% tumor formation each) compared with 100% tumor formation of C3L5 cells expressing only neomycin phosphotransferase (C3L5-G1N). However, when tumor-free animals were challenged with parental C3L5 cells, C3L5-Flt3L vaccination was significantly better at preventing tumor growth (p < 0.05) than C3L5-GMCSF vaccination (33% of C3L5-Flt3L-vaccinated animals developed tumor compared with 77% of C3L5-GMCSF-vaccinated animals). Adoptive transfer of immunity for both vaccines was demonstrated; splenic T cells from tumor-free mice protected naive mice from parental tumor challenge. To simulate minimal disease, parental C3L5 cells at two concentrations (high, 5 X 10 3 cells; or low, 1 X 10 3 cells) were injected into the contralateral chest wall 4 days prior to treatment with C3L5-G1N or C3L5-Flt3L. C3L5-Flt3L treatment decreased contralateral parental tumor formation (high, 67% tumor free; low, 90% tumor free) compared with C3L5-G1N treatment (high and low, 0% tumor free). Immunodepletion of activated natural killer cells with anti-asialo-GM1 blocked C3L5-Flt3L- and C3L5 plus soluble Flt3L-mediated antitumor activity. Thus, Flt3L-transduced tumor cells manifest potent antitumor activity, apparently mediated, at least partially, by natural killer cells.

We have previously reported that intravenous administration of cationic lipid-protamine-DNA complexes (LPD) induces production of large quantities of proinflammatory cytokines that are toxic and cause inhibition of transgene expression. Cytokine induction appears to be mediated by the unmethylated CpG sequences since methylation of plasmid DNA significantly decreases the cytokine levels. In this study, the inhibitory role of CpG in lipid-mediated gene transfer was further investigated using chemically well-defined, CpG-containing oligodeoxynucleotides (ODNs). Injection (intravenous) of ODNs formulated in LPD into mice triggered production of proinflammatory cytokines including interferon gamma and TNF-alpha. The potency of CpG-containing ODNs in cytokine induction was affected by its flanking sequences and was significantly reduced when CpG was methylated. Preinjection of ODN-containing LPD led to inhibition of transgene expression in lungs after a subsequent injection of LPD containing plasmid expression vector with luciferase gene. The degree of inhibition correlated with the levels of ODN-triggered cytokines. Finally, intraperitoneal injection of dexamethasone suppressed LPD-induced cytokine production, and led to significantly higher levels of transgene expression on both first and second injection. These studies suggest that mutation of potent CpG motifs in plasmid DNA together with the use of immune suppression agent may represent an effective approach to improve cationic lipid-mediated gene transfer to the lung.

Mucopolysaccharidosis type I (MPS I), a deficiency in the lysosomal enzyme alpha-L-iduronidase (IDUA), is characterized by skeletal abnormalities, hepatosplenomegaly and neurological dysfunction. To evaluate the potential for treatment of the disease using a gene delivery approach, recombinant adeno-associated virus (rAAV) vectors were constructed and evaluated for expression of the human IDUA cDNA in transduced cells. 293 cells transduced with these AAV vectors contained IDUA activity at 0.5 to 1.4 mu mol/mg.hr, 50- to 140-fold above background (control-transduced) levels. In time course studies of transduced 293 cells, IDUA activity levels peaked 1 week after transduction and persisted at 50% of the peak level for at least 6 weeks. Transduced MPS I fibroblasts also expressed high levels of IDUA activity (114-290 nmol/mg.hr), which persisted for at least 3 weeks in the absence of selection. In addition, transduced MPS I fibroblasts were capable of clearing intracellular radiolabeled glycosaminoglycan (GAG). As a test of the ability of these vectors to mediate metabolic cross-correction, transduced HuH7 human hepatoma cells were demonstrated to release enzyme that was subsequently taken up by nontransduced MPS I fibroblasts. These results illustrate the effectiveness of AAV vectors for delivery and expression of human IDUA gene sequences and for potential treatment of MPS I.

Protection of hematopoietic cells of patients undergoing anticancer chemotherapy by MDR1 gene transfer is currently being studied in clinical trials. From animal studies, it has been suggested that aberrant splicing due to cryptic donor and acceptor sites in the MDR1 cDNA could be a major reason for failure to obtain high-level expression of P-glycoprotein in bone marrow. We investigated effects of drug selection on protein expression levels and on splicing of MDR1 transcripts in murine bone marrow cells (BMCs) in vitro . To this end, retroviruses were generated through an identical plasmid, pHaMDR1/A, introduced into different packaging cells. GP + E86- but not PA317-derived producer cells were found to express truncated in addition to full-length message. In BMCs transduced with GP + E86-derived viruses, both messages were increased after treatment with colchicine or daunomycin. Similar results were obtained with NIH 3T3 fibroblasts. However, transduced and drug-selected BMCs displayed the spliced transcript even if the respective PA317-derived producer cells contained no truncated RNA as detected in transduced NIH 3T3 fibroblasts. Short-term drug selection in BMCs transduced with either ecotropic or amphotropic retroviruses resulted in a striking increase in P-glycoprotein expression. Thus, aberrant splicing failed to abrogate P-glycoprotein expression in BMCs. We also studied a vector in which MDR1 was coexpressed with glucocerebrosidase, using an internal ribosomal entry site. Although chemoprotection was less efficient than with pHaMDR1/A, augmentation of protein expression was observed at low selecting drug concentrations. Our study shows that drug selection can partially compensate for inefficient transduction of hematopoietic cells, and may help to develop strategies by which unstable expression of transduced genes can be overcome.

Adenovirus (Ad) vectors used for gene therapy are efficient in entering the infected cell and targeting their genome to the nucleus. To study the mechanism of the interaction between Ad and the nuclear envelope we have established an in vitro assay using rat liver nuclei incubated with serotype 5 Ad vector. Binding of either fluorophore (Cy3)-labeled Ad5 (Cy3-AdGFP) or [3H]Ad5 was blocked by excess unlabeled Ad5, indicating that the interaction was specific. Binding reached equilibrium within 30 min, exhibited temperature dependence with more binding occurring at 37 degrees C than at 4 degrees C and appeared to be irreversible. Prior treatment of nuclei with glutaraldehyde or proteolysis of nuclei with trypsin inhibited the Cy3-AdGFP association with nuclei, and pretreatment of Cy3-AdGFP with human anti-Ad5 serum, but not naive human serum, inhibited Cy3-AdGFP, suggesting a requirement for direct interaction between Ad5 and nuclei. Addition of excess unlabeled Ad serotype 2 or Ad serotype 7 competed for binding with Cy3-AdGFP, indicating that the capsid determinant of nuclear binding was conserved among group B and C Ad serotypes. These data suggest that the Ad capsid and nuclear envelope contain specific domains that mediate binding of the two entities and that binding mechanisms to the nuclear envelope might be a common final pathway of different Ad serotypes.

A high therapeutic index is as important for gene-based therapies as it is for chemotherapy or radiotherapy. One approach has been transcriptional targeting through the use of tissue-specific regulatory elements. A more versatile approach would be to use a regulatory element that is controlled via a parameter common to a broad range of diseases. Ischemia is characteristic of a number of pathologies that range from vascular occlusion through to cancer. The state of low oxygen, hypoxia, triggers a transcriptional signaling pathway that is mediated by transcription factors binding to a specific enhancer, the hypoxia response element (HRE). These observations have therefore led to the use of HREs to drive gene expression in a number of target tissues from tumors to cardiac muscle. To translate these observations into a clinically useful vector system we have now assessed the potency of a number of naturally derived HREs in various configurations combined with minimal promoters. The optimal HRE has been introduced into a single transcription unit retroviral vector that can deliver regulated gene expression in response to hypoxia. An important feature of this new physiologically regulated vector is the combination of low basal expression and high-level activated expression that is on a par with that obtained with the cytomegalovirus immediate-early (CMV IE) promoter. The role of elements that stabilize mRNA in the presence of hypoxia has also been assessed. These hypoxia-regulated vectors may have utility for restricting the delivery of therapeutic proteins to tumors and ischemic sites.

Genetic diseases are often caused by nonsense mutations. The resulting defect in protein translation can be restored by expressing suppressor tRNA in the mutant cells. Our goal was to demonstrate both protein restoration and phenotypic correction using these small transgenes. Functional activity of an arginine opal suppressor tRNA in cells expressing a nonsense mutated GFP gene was demonstrated by restored fluorescence. This suppressor tRNA was expressed in xeroderma pigmentosum group A cells, containing a homozygous nonsense mutation at Arg-207 in the XPA complementing gene. The transfected XPA cell population showed a twofold increase in cell survival after UV irradiation as determined by colony-forming assays compared with cell populations without the suppressor tRNA gene. The UV doses required for 37% survival of XP cells and XP cells expressing the suppressor tRNA were 0.6 and 1.2 J/m2. A similar twofold increase in the reactivation of UV-irradiated plasmid DNA was observed in XP cells expressing the suppressor tRNA. However, there was no detectable increase in XPA protein levels. Several potential limitations of this approach exist, including the availability of mutant RNA transcripts, the efficiency of suppression by the suppressor tRNA, and the abundance and availability and continued expression of the suppressor tRNA. The unique feature of this study is the relatively small size (88 bp) of the suppressor tRNA. Small-sized suppressor tRNAs can be synthetically constructed and subcloned into different viral vectors for delivery into the target cells. This approach may be useful for other genetic diseases caused by nonsense mutations.

Primary human T lymphocytes were transduced at high efficiency with the Moloney murine leukemia virus (Mo-MuLV) vector, LNC-mB7-1, in which an internal cytomegalovirus (CMV) promoter drives expression of the murine B7-1 cDNA. Compared with transduced T cells expanded in IL-2 or reactivated with soluble antibodies to CD3 or CD28, transgene expression was significantly increased after activation on immobilized anti-CD3 antibodies (CD3i) or by simultaneous activation on immobilized anti-CD3 and anti-CD28 antibodies (CD3i/CD28i). A similar pattern of transgene expression was observed in T cells transduced with MoMuLV LNC-EGFP. Proviral copy number was maintained in LNC-mB7-1-transduced T cells expanded in IL-2 or reactivated on CD3i/CD28i. Substantial increases in LNC-mB7-1 steady state mRNA in reactivated T lymphocytes, compared with those maintained in IL-2, correlated with increased transcription of the LNC-mB7-1 proviral DNA. Furthermore, T cells transduced with the Mo-MuLV ZIPPGK-mADA, in which the mADA cDNA is driven by an internal human phosphoglycerate kinase (PGK) promoter, showed increases in steady state ZIPPGK-mADA RNA on reactivation. High levels of transgene expression were evident irrespective of cell cycle position in both CD4+ and CD8+ lymphocytes. After reactivation, increases in LNC-mB7-1 mRNA were observed in the presence of the protein synthesis inhibitor cycloheximide, indicating that proteins involved in upregulating transgene expression preexisted in transduced lymphocytes. Induction of transgene expression on CD3i/CD28i showed a dose-dependent decrease in transgene expression when incubated with selective protein kinase inhibitors. These data provide new insights into the mechanisms governing transgene expression driven by Mo-MuLV constructs containing internal promoters in transduced primary T lymphocytes.

Prostate adenocarcinoma is the most common nonskin malignancy in males and the second most common cause of cancer death in the United States (Landis et al., 1998). Initial treatments of surgery or radiotherapy may cause impotence and/or incontinence from neural damage (Eastham and Scardino, 1998; Porter et al., 1998). When extraprostatic or metastatic disease develops, castration or pharmaceutical androgen ablation is utilized (Catalona, 1994). Androgen-resistant recurrence indicates a poor prognosis and justifies experimental chemotherapy (Oh and Kantoff, 1998). G207 (Mineta et al., 1995; Yazaki et al., 1995) is a multimutated herpes simplex virus 1 (HSV) vector that replicates within cancer cells, causing cellular death; however, replication is limited in normal cells, including those of the nervous system. In vitro, G207 at a low multiplicity of infection (MOI of 0.01) is oncolytic for multiple human prostate cancer cells. In athymic mice, a single intraneoplastic inoculation of G207 completely eradicates >22% of established subcutaneous human prostate cancer tumors irrespective of hormonal responsiveness. Two intraneoplastic inoculations of G207 completely eradicated two of three recurrent previously irradiated tumors and two intravenous administration of G207 induced tumor regression in distant subcutaneous tumors and completely eradicated one-fourth of the tumors.

Bone morphogenetic proteins (BMPs) are polypeptides that induce ectopic bone formation in standard rat in vivo assay systems. Previous studies have demonstrated the clinical utility of these proteins in spinal fusion, fracture healing, and prosthetic joint stabilization. Gene therapy is also a theoretically attractive technique to express BMPs clinically, since long-term, regulatable gene expression and systemic delivery with tissue-specific expression may be possible in future. This study was performed to determine whether an adenoviral vector containing the BMP-2 gene can be used to express BMP-2 in vitro and promote endochondral bone formation in vivo. In vitro, U87 MG cells transduced per cell with 20 MOI of an adenoviral construct containing the BMP-2 gene under the control of the universal CMV promoter (Ad-BMP-2) showed positive antibody staining for the BMP-2 protein at posttransfection day 2. The synthesis and secretion of active BMP-2 into the conditioned medium of Ad-BMP-2-transduced 293 cells were confirmed by Western blot analysis and the induction of alkaline phosphatase activity in a W-20 stromal cell assay. In vivo, Sprague-Dawley rats and athymic nude rats were injected with Ad-BMP-2 in the thigh musculature and were sacrificed on day 3, 6, 9, 12, 16, 21, 60, and 110 for histological analysis. The Sprague-Dawley rats showed evidence of acute inflammation, without ectopic bone formation, at the injection sites. In the athymic nude rats, BMP-2 gene therapy induced mesenchymal stem cell chemotaxis and proliferation, with subsequent differentiation to chondrocytes. The chondrocytes secreted a cartilaginous matrix, which then mineralized and was replaced by mature bone. This study demonstrates that a BMP-2 adenoviral vector can be utilized to produce BMP-2 by striated muscle cells in athymic nude rats, leading to endochondral bone formation. However, in immunocompetent animals the endochondral response is attenuated, secondary to the massive immune response elicited by the first-generation adenoviral construct.