Gene therapy

In recent years, there have been a number of technological breakthroughs that have allowed for clinical trials in gene therapy to be initiated. In combination with the genome initiative, the potential for new therapeutics is limitless. Although an enormous amount of information has been obtained in a relatively short period of time, gene therapy is not yet ready for wide-scale practice. Some of the successes and obstacles that remain are summarized in this report.

Adenovirus-mediated interleukin-12 gene therapy for metastatic colon carcinoma.

Recombinant adenoviral mediated delivery of suicide and cytokine genes has been investigated as a treatment for hepatic metastases of colon carcinoma in mice. Liver tumors were established by intrahepatic implantation of a poorly immunogenic colon carcinoma cell line (MCA-26), which is syngeneic in BALB/c mice. Intratumoral transfer of the herpes simplex virus type 1 thymidine kinase (HSV-tk) and the murine interleukin (mIL)-2 genes resulted in substantial hepatic tumor regression, induced an effective systemic antitumoral immunity in the host and prolonged the median survival time of the treated animals from 22 to 35 days. The antitumoral immunity declined gradually, which led to tumor recurrence over time. A recombinant adenovirus expressing the mIL-12 gene was constructed and tested in the MCA-26 tumor model. Intratumoral administration of this cytokine vector alone increased significantly survival time of the animals with 25% of the treated animals still living over 70 days. These data indicate that local expression of IL-12 may also be an attractive treatment strategy for metastatic colon carcinoma.

Development of HIV vectors for anti-HIV gene therapy.

Current gene therapy protocols for HIV infection use transfection or murine retrovirus mediated transfer of antiviral genes into CD4+ T cells or CD34+ progenitor cells ex vivo, followed by infusion of the gene altered cells into autologous or syngeneic/allogeneic recipients. While these studies are essential for safety and feasibility testing, several limitations remain: long-term reconstitution of the immune system is not effected for lack of access to the macrophage reservoir or the pluripotent stem cell population, which is usually quiescent, and ex vivo manipulation of the target cells will be too expensive and impractical for global application. In these regards, the lentivirus-specific biologic properties of the HIVs, which underlie their pathogenetic mechanisms, are also advantageous as vectors for gene therapy. The ability of HIV to specifically target CD4+ cells, as well as non-cycling cells, makes it a promising candidate for in vivo gene transfer vector on one hand, and for transduction of non-cycling stem cells on the other. Here we report the use of replication-defective vectors and stable vector packaging cell lines derived from both HIV-1 and HIV-2. Both HIV envelopes and vesicular stomatitis virus glycoprotein G were effective in mediating high-titer gene transfer, and an HIV-2 vector could be cross-packaged by HIV-1. Both HIV-1 and HIV-2 vectors were able to transduce primary human macrophages, a property not shared by murine retroviruses. Vesicular stomatitis virus glycoprotein G-pseudotyped HIV vectors have the potential to mediate gene transfer into non-cycling hematopoietic stem cells. If so, HIV or other lentivirus-based vectors will have applications beyond HIV infection.

Fusigenic viral liposome for gene therapy in cardiovascular diseases.

To improve the efficiency of liposome-mediated DNA transfer as a tool for gene therapy, we have developed a fusigenic liposome vector based on principles of viral cell fusion. The fusion proteins of hemagglutinating virus of Japan (HVJ; also Sendai virus) are complexed with liposomes that encapsulate oligodeoxynucleotide or plasmid DNA. Subsequent fusion of HVJ-liposomes with plasma membranes introduces the DNA directly into the cytoplasm. In addition, a DNA-binding nuclear protein is incorporated into the HVJ-liposome particle to enhance plasmid transgene expression. The fusigenic viral liposome vector has proven to be efficient for the intracellular introduction of oligodeoxynucleotide, as well as intact genes up to 100 kbp, both in vitro and in vivo. Many animal tissues have been found to be suitable targets for fusigenic viral liposome DNA transfer. In the cardiovascular system, we have documented successful cytostatic gene therapy in models of vascular proliferative disease using antisense oligodeoxynucleotides against cell cycle genes, double-stranded oligodeoxynucleotides as "decoys" to trap the transcription factor E2F, and expression of a transgene encoding the constitutive endothelial cell form of nitric oxide synthase. Similar strategies are also effective for the genetic engineering of vein grafts and for the treatment of a mouse model of immune-mediated glomerular disease.

Gene gun-mediated skin transfection with interleukin 12 gene results in regression of established primary and metastatic murine tumors.

Particle-mediated (gene gun) in vivo delivery of the murine interleukin 12 (IL-12) gene in an expression plasmid was evaluated for antitumor activity. Transfer of IL-12 cDNA into epidermal cells overlying an implanted intradermal tumor resulted in detectable levels (266.0 +/- 27.8 pg) of the transgenic protein at the skin tissue treatment site. Despite these low levels of transgenic IL-12, complete regression of established tumors (0.4-0.8 cm in diameter) was achieved in mice bearing Renca, MethA, SA-1, or L5178Y syngeneic tumors. Only one to four treatments with IL-12 cDNA-coated particles, starting on day 7 after tumor cell implantation, were required to achieve complete tumor regression. This antitumor effect was CD8+ T cell-dependent and led to the generation of tumor-specific immunological memory. By using a metastatic P815 tumor model, we further showed that a delivery of IL-12 cDNA into the skin overlying an advanced intradermal tumor, followed by tumor excision and three additional IL-12 gene transfections, could significantly inhibit systemic metastases, resulting in extended survival of test mice. These results suggest that gene gun-mediated in vivo delivery of IL-12 cDNA should be further developed for potential clinical testing as an approach for human cancer gene therapy.

Plasmoviruses: nonviral/viral vectors for gene therapy.

We have generated a chimeric gene transfer vector that combines the simplicity of plasmids with the infectivity and long-term expression of retroviruses. We replaced the env gene of a Moloney murine leukemia virus-derived provirus by a foreign gene, generating a plasmid that upon transfer to tumor cells generates noninfectious retroviral particles carrying the transgene. We added to this plasmid an independent expression cassette comprising a cytomegalovirus promoter, an amphotropic retroviral envelope, and a polyadenylylation signal from simian virus 40. These constructs were designed to minimize the risk of recombination generating replication-competent retroviruses. Their only region of homology is a 157-bp sequence with 53% identity. We show that the sole transfection of this plasmid in various cell lines generates infectious but defective retroviral particles capable of efficiently infecting and expressing the transgene. The formation of infectious particles allows the transgene propagation in vitro. Eight days after transfection in vitro, the proportion of cells expressing the transgene is increased by 10-60 times. There was no evidence of replication-competent retrovirus generation in these experiments. The intratumoral injection of this plasmid, but not of the control vector lacking the env gene, led to foci of transgene-expressing cells, suggesting that the transgene had propagated in situ. Altogether, these "plasmoviruses" combine advantages of viral and non-viral vectors. They should be easy to produce in large quantity as clinical grade materials and should allow efficient and safe in situ targeting of tumor cells.

Human immunodeficiency virus (HIV) type 2-mediated inhibition of HIV type 1: a new approach to gene therapy of HIV-infection.

Human immunodeficiency virus (HIV) type 2, the second AIDS-associated human retrovirus, differs from HIV-1 in its natural history, infectivity, and pathogenicity, as well as in details of its genomic structure and molecular behavior. We report here that HIV-2 inhibits the replication of HIV-1 at the molecular level. This inhibition was selective, dose-dependent, and nonreciprocal. The closely related simian immunodeficiency provirus also inhibited HIV-1. The selectivity of inhibition was shown by the observation that HIV-2 did not significantly downmodulate the expression of the unrelated murine leukemia virus; neither did the murine leukemia virus markedly affect HIV-1 or HIV-2 expression. Moreover, while HIV-2 potently inhibited HIV-1, the reverse did not happen, thus identifying yet another and remarkable difference between HIV-1 and HIV-2. Mutational analysis of the HIV-2 genome suggested that the inhibition follows a complex pathway, possibly involving multiple genes and redundant mechanisms. Introduction of inactivating mutations into the structural and regulatory/accessory genes did not render the HIV-2 provirus ineffective. Some of the HIV-2 gene defects, such as that of tat and rev genes, were phenotypically transcomplemented by HIV-1. The HIV-2 proviruses with deletions in the putative packaging signal and defective for virus replication were effective in inducing the suppressive phenotype. Though the exact mechanism remains to be defined, the inhibition appeared to be mainly due to an intracellular molecular event because it could not be explained solely on the basis of cell surface receptor mediated interference. The results support the notion that the inhibition likely occurred at the level of viral RNA, possibly involving competition between viral RNAs for some transcriptional factor essential for virus replication. Induction of a cytokine is another possibility. These findings might be relevant to the clinical-epidemiological data suggesting that infection with HIV-2 may offer some protection against HIV-1 infection.

Eradication of established intracranial rat gliomas by transforming growth factor beta antisense gene therapy.

Like human gliomas, the rat 9L gliosarcoma secretes the immunosuppressive transforming growth factor beta (TGF-beta). Using the 9L model, we tested our hypothesis that genetic modification of glioma cells to block TGF-beta expression may enhance their immunogenicity and make them more suitable for active tumor immunotherapy. Subcutaneous immunizations of tumor-bearing animals with 9L cells genetically modified to inhibit TGF-beta expression with an antisense plasmid vector resulted in a significantly higher number of animals surviving for 12 weeks (11/11, 100%) compared to immunizations with control vector-modified 9L cells (2/15, 13%) or 9L cells transduced with an interleukin 2 retroviral vector (3/10, 30%) (P < 0.001 for both comparisons). Histologic evaluation of implantation sites 12 weeks after treatment revealed no evidence of residual tumor. In vitro tumor cytotoxicity assays with lymph node effector cells revealed a 3- to 4-fold increase in lytic activity for the animals immunized with TGF-beta antisense-modified tumor cells compared to immunizations with control vector or interleukin 2 gene-modified tumor cells. These results indicate that inhibition of TGF-beta expression significantly enhances tumor-cell immunogenicity and supports future clinical evaluation of TGF-beta antisense gene therapy for TGF-beta-expressing tumors.

Systemic gene therapy: biodistribution and long-term expression of a transgene in mice.

We have investigated the in vivo efficacy of a systemic gene transfer method, which combines a liposomal delivery system (DLS liposomes) with episomally replicative DNA plasmids to effect long-term expression of a transgene in cells. A single i.v. injection of a plasmid DNA vector containing the luciferase gene as a marker was administered with the DLS liposomes in BALB/c mice. The luciferase gene and its product were found in all mouse tissues tested as determined by PCR analysis and immunohistochemistry. Luciferase activity was also detected in all tissues tested and was present in lung, liver, spleen, and heart up to 3 months postinjection. In contrast to the nonepisomal vectors tested (pRSV-luc and pCMVintlux), human papovavirus (BKV)-derived episomal vectors showed long-term transgene expression. We found that these episomal vectors replicated extrachromosomally in lung 2 weeks postinjection. Results indicated that transgene expression in specific tissues depended on the promoter element used, DNA/liposome formulation, dose of DNA per injection, and route of administration.

A gene therapy strategy using a transcription factor decoy of the E2F binding site inhibits smooth muscle proliferation in vivo.

The application of DNA technology to regulate the transcription of disease-related genes in vivo has important therapeutic potentials. The transcription factor E2F plays a pivotal role in the coordinated transactivation of cell cycle-regulatory genes such as c-myc, cdc2, and the gene encoding proliferating-cell nuclear antigen (PCNA) that are involved in lesion formation after vascular injury. We hypothesized that double-stranded DNA with high affinity for E2F may be introduced in vivo as a decoy to bind E2F and block the activation of genes mediating cell cycle progression and intimal hyperplasia after vascular injury. Gel mobility-shift assays showed complete competition for E2F binding protein by the E2F decoy. Transfection with E2F decoy inhibited expression of c-myc, cdc2, and the PCNA gene as well as vascular smooth muscle cell proliferation both in vitro and in the in vivo model of rat carotid injury. Furthermore, 2 weeks after in vivo transfection, neointimal formation was significantly prevented by the E2F decoy, and this inhibition continued up to 8 weeks after a single transfection in a dose-dependent manner. Transfer of an E2F decoy can therefore modulate gene expression and inhibit smooth muscle proliferation and vascular lesion formation in vivo.