Current Gene Therapy - Volume 2, Issue 1, 2002

One of the main barriers to more efficacious use of modern chemotherapeutic agents, is the collateral toxicity exhibited in normal, highly proliferative tissues, primarily the haemopoietic, gastrointestinal and Pulmonary tissues. Drug resistance of tumours to these drugs compounds this problem. This review discusses the role of O6-alkylguanine-DNA alkyltransferase (ATase) in conferring protection against O6-alkylating agents in normal tissue, focusing mainly on the haemopoietic compartment. The development of mutant forms of ATase, which are resistant to the effects of soluble analogues of O6-alkylation such as O6-benzylguanine, is examined and the gene therapy approach of combining these two strategies to confer chemoprotection to vulnerable tissues whilst sensitising malignant tissue is reviewed.

Transplantation offers a unique opportunity for gene transfer into allografts before grafting. After organ retrieval, the cold ischemic period renders organs available for manipulation and gene transfer. Local expression of protective or immunomodulatory molecules within the graft environment offers a better local bioavailability of bioreagents and potentially less systemic side effects. Protection against ischemia-reperfusion injury, acute and / or chronic rejection without significant side effects would be a major breakthrough in transplant research. However, protocols of transfection adapted to the transplant setting and control of gene expression must be clearly evaluated before going to clinical trials. The first part of this review deals with gene transfer techniques into the allograft, emphasizing particular transplant conditions that are encountered and that must be respected when designing protocols for gene transfer experiments. The second part deals with specific therapeutic strategies to protect and prolong allograft survival.

Lentiviral vectors based on HIV-1, HIV-2, or SIV have the ability to transduce dividing and non-dividing T cells, dendritic cells, hematopoietic stem cells and macrophages, which are the main target cells for gene therapy of HIV-1 infection. Besides their function as gene delivery vehicles, lentiviral vector backbones containing the cis-acting sequences necessary to perform a complete replication cycle in the presence of viral proteins provided in trans, have the ability to inhibit HIV-1 replication by several mechanisms that include sequestration of the regulatory proteins Tat and Rev, competition for packaging into virions and possibly by inhibition of reverse transcription in heterodimeric virions. Expression of anti-HIV-1 genes in these vectors would strengthen the potency of this inhibition. To avoid self-inhibition of the vector packaging system, lentiviral vectors have to be modified to become resistant to the anti-HIV-1 genes encoded by them. This review discusses the different genetic intervention strategies for gene therapy of HIV-1 infection focusing in the use of lentiviral vectors as the main agents to mediate inhibition of HIV-1 replication. It also discusses possible strategies to adapt HIV-1 or HIV-2 vectors to express the different classes of anti-HIV-1 genes and approaches to improve in vivo vector mobilization

Gene transfer into hematopoietic cells is currently being used to modulate immune responses, to protect hematopoietic cells against cytotoxic drugs or viral genes, and to restore gene deficiencies due to either inborn genetic defects or acquired loss of regular gene function. In particular, gene addition strategies for inherited severe combined immunodeficiencies (SCID) due to adenosine deaminase (ADA) deficiency or defects of the interleukin-2 receptor γ-chain represent potentially curative strategies based on gene transfer into hematopoietic cells using recombinant retroviral vectors. Since long-term correction of genetic defects in hematopoietic cells often requires transduction of hematopoietic stem cells, an effective gene transfer into stem cells with efficient long-term and multi-lineage transgene expression is the desired goal for these therapeutic strategies. However, gene transfer strategies with retroviral vectors unable to integrate into non-cycling cells are limited by the quiescent state of the stem cells that have to be stimulated by cytokines to induce cell cycle progression. To circumvent these barriers, lentiviral vector systems based on HIV-1 have recently been developed which are able to deliver and express genes in non-dividing cells both in vitro and in vivo. This review outlines the development and improvement of lentivirus-based gene transfer protocols and discusses the use of lentiviral vectors in preclinical gene therapy studies.

Gene-engineered dendritic cells (DC) are being tested in cancer immunotherapy. DC are the best equipped antigen-presenting cells (APC) to overcome tolerance / ignorance to self antigens presented by cancer cells. Genetic immunotherapy with DC engineered to express tumor antigens has the potential advantages of endogenous epitope presentation by both major histocompatibility complex (MHC) class I and II molecules. DC can also be gene-modified to express immunostimulatory molecules that further enhance their antigen-presenting function. Review of the literature provided 52 manuscripts where gene-modified DC were being tested in murine models of immunotherapy for cancer. Review of the antitumor effects of gene-modified DC in these preclinical studies provides valuable information on the optimal methods of gene transfer into DC, the schedule of administration, the route, dose and the underlying immunological mechanisms of the antitumor effects. These data may help in the translation of this promising approach to the clinic.

Bone marrow-derived dendritic cells have been used to treat established experimental tumors by unleashing a cellular immune response against tumor antigens. Such antigens are artificially loaded onto dendritic cells' antigenpresenting molecules by different techniques including incubation with synthetic antigenic determinants, tumor lysates or nucleic acids encoding for those relevant antigens. Ex vivo gene transfer with viral and non-viral vectors is frequently used to obtain expression of the tumor antigens and thereby to formulate the therapeutic vaccines. Efficacy of the approaches is greatly enhanced if dendritic cells are transfected with a number of genes which encode immunostimulating factors. In some cases, such as with IL-12, IL-7 and CD40L genes, injection inside experimental malignancies of thus transfected dendritic cells induces complete tumor regression in several models. In this case tumor antigens are captured by dendritic cells by still unclear mechanisms and transported to lymphoid organs where productive antigen presentation to T-cells takes place. Many clinical trials testing dendritic cell-based vaccines against cancer are in progress and partial clinical efficacy has been already proved. Transfection of genes further strengthening the immunogenicity of such strategies will join the clinical club soon.

A variety of adoptive cellular strategies, aimed at boosting the immune system, have been tested in the management of metastatic diseases. Despite the drawbacks associated with ex vivo cell manipulation and upscaling, several such approaches have been assessed in the clinic. The use of lymphokine-activated killer (LAK) cells, autolymphocyte therapy (ALT) and tumor-infiltrating lymphocytes (TIL) have been the best studied and further trials are ongoing. Thus far, these approaches have not consistently shown benefit when compared to standard immune-based treatment with biologic response modifiers, notably, high-dose interleukin-2 (IL-2). More recently, it has been shown, in various animal models, that the ex vivo transfer of genes to cells of the immune system can have a dramatic impact on cancer immunotherapy. The application of gene transfer techniques to immunotherapy has animated the field of cell-based cancer therapy research. A wide variety of viral and non-viral gene transfer methods have been investigated in this context. Ex vivo strategies include gene delivery into tumor cells and into cellular components of the immune system, including cytotoxic T cells, NK, macrophages and dendritic cells (DC). Several of these approaches have already been translated into cancer therapy clinical trials. In this review, we focus on the rationale and types of ex vivo gene-based immunotherapy of cancer. Finally, the use of genetically modified DC for tumor vaccination and its prospects are discussed.

Muscle is a convenient and accessible site for non-viral gene delivery, which can manufacture gene products and provide a long-duration of gene expression. The level of gene expression after administration of naked DNA plasmid or polymer-formulated DNA plasmid containing a reporter gene to muscle via syringe injection, however, is very low. As a result, no significant therapeutic effect can be detected after saline- or polymer-mediated gene delivery into muscle. In 1998, investigators published a striking new approach-electrotransfection-for intramuscular gene delivery (now commonly referred to as electroporation or electroinjection). Electroporation of a non-viral gene into the muscles of small animals has increased the level of gene expression by as much as two orders of magnitude, which is comparable to levels achieved with adenoviral gene delivery. Three years later, intramuscular electroporation gene delivery technology has blossomed. Treatments for different diseases using this approach in animal models have been reported. In this review, I discuss the applications of intramuscular electroporation gene therapy to treat malignancies, renal disease, and anemia, and to prevent drug toxicity to sensory nerves.