Gene Therapy For Neoplastic And Infectious Diseases

· Gene Therapy
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Cancer gene therapies

Many different approaches can be used for cancer gene therapy and, in marked contrast to the few gene therapy trials for inherited disorders, numerous cancer gene therapy trials are currently being conducted. This reflects partly the severity of the disorders that are being treated and the considerable funding for cancer research, and partly reflects the comparative ease in applying treatments based on targeted killing of disease cells, by introducing genes that encode toxins, etc. or by provoking enhanced immune responses. In a few cases, the gene therapy approach has focused on targeting single genes, such as TP53 gene augmentation therapy and delivery of antisense KRAS genes in the case of some forms of non-small-cell lung cancer. In most cases, however, targeted killing of cancer cells has been conducted without knowing the molecular etiology of the cancer. Thus far, some significant advances have been made against local and metastatic tumor growth, but effective therapy awaits development of more effective methods to transfer and express transgenes or to induce antitumor responses

General approaches

Artificial killing of cancer cells

  • Insert a gene encoding a toxin (e.g. diphtheria A chain) or a gene conferring sensitivity to a drug (e.g. herpes simplex thymidine kinase) into tumor cells
  • Stimulate natural killing of cancer cells
  • Enhance the immunogenicity of the tumor by, for example, inserting genes encoding foreign antigens or cytokines
  • Increase antitumor activity of immune system cells by, for example, inserting genes that encode cytokines
  • Induce normal tissues to produce antitumor substances (e.g. interleukin-2, interferon)
  • Production of recombinant vaccines for the prevention and treatment of malignancy (e.g. BCG-expressing tumor antigens)
  • Protect surrounding normal tissues from effects of chemotherapy/radiotherapy
  • Protect tissues from the systemic toxicities of chemotherapy (e.g. multiple drug resistance type 1 gene)

Tumors resulting from oncogene activation

  • Selectively inhibit the expression of the oncogene
  • Deliver gene-specific antisense oligonucleotide or ribozyme to bind/cleave oncogene mRNA
  • Inhibit transcription by triple helix formation following delivery of a gene-specific oligonucleotide
  • Use of intracellular antibodies or oligonucleotide aptamers to specifically bind to and inactivate the oncoprotein

Tumors arising from inactivation of tumor suppressor

  • Gene augmentation therapy
  • Insert wild-type tumor suppressor gene 

Ex vivo cancer gene therapies for cancer cells

Ex vivo cancer gene therapies frequently involve attempts to recruit immune system cells to destroy the tumor cells

Gene transfer into tumor-infiltrating lymphocytes

One of the earliest gene therapy protocols used a population of immune system cells for specifically targeting a foreign protein to a tumor. The therapy could be considered to be a form of adoptive immunotherapy (see below) because a gene encoding a cytokine, tumor necrosis factor-α (TNF-α), was transferred into tumor-infiltrating lymphocytes (TILs) in an effort to increase their antitumor efficacy. The TIL population is a natural population of T lymphocytes which can seek out and infiltrate tumor deposits, such as metastatic melanomas. TNF-α is a protein naturally produced by T lymphocytes which, if infused in sufficient amounts in mice, can destroy tumors. However, it is a toxic substance and intravenous infusion of TNF has significant adverse side-effects in humans. An attractive alternative was to use TILs as cellular vectors for transferring the toxic protein directly to tumors. The gene therapy approach that was used, therefore, involved retroviral-mediated transfer of a TNF gene to a TIL population which had initially been obtained from an excised tumor and then grown in culture. Subsequent transfusion of the genetically modified TILs into a patient with metastatic melanoma was expected to result in the TILs ‘homing in’ on the melanomas, expression of the introduced TNF gene and tumor regression. However, the trial has been marked by comparatively poor efficiency of gene transfer into human TILs and a down-regulation of cytokine expression by the TILs.

 This approach has been used in an attempt at ex vivo gene therapy for metastatic melanoma. The tumor-infiltrating lymphocytes (TIL) appear to be able to ‘home in’ to tumor deposits. In this example, they act as cellular vectors for transporting to the melanomas a retrovirus recombinant which contains a gene specifying the anti-tumor cytokine TNF-α (tumor necrosis factor-α). Problems with the efficiency of gene transfer into the TILs and down-regulation of cytokines limited the success of this approach.

Adoptive immunotherapy by genetic modification of tumor cells

Animal studies in which murine tumor cells were genetically modified by the insertion of genes encoding various cytokines [several different interleukins (ILs), TNF-α, interferon (IFN)-γ, granulocyte—macrophage colonystimulating factor (GM-CSF)] and then re-implanted in mice gave cause for encouragement. In each case, the genetically altered tumor cells either never grew, or grew and then regressed. In addition, most of the treated mice were then systemically immune to reimplantation of nonmodified tumors. However, the results were much less satisfactory when animals with established, sizeable tumors were treated. Nevertheless, the idea of modifying a patient’s own tumor cells for use as a vaccine (adoptive immunotherapy) caught on, and human gene therapy trials have been approved for the insertion of cytokine genes using retrovirus vectors for treating a wide variety of cancers.

In each case, the idea is to immunize the patients specifically against their own tumors by genetically modifying the tumor with one of a variety of genes that are expected to increase the host immune reactivity to the tumor. In addition to cytokine genes, other genes such as foreign HLA antigen genes have been transferred to tumors for the same general reason. Insertion of genes encoding HLA-B7 into tumors of patients lacking HLA-B7 is intended to provoke an immune response to the tumors as a consequence of the presence on the tumor cell surface of the effectively foreign HLA-B7 antigen. Such a response is hoped to provide subsequent immunity against the same type of tumor even in the absence of the HLA-B7 antigen.

Adoptive immunotherapy by genetic modification of fibroblasts

One problem with ex vivo therapy for tumors is the difficulty in growing tumor cells in vitro: less than 50% of tumor cell lines grow in long-term culture. As an alternative, fibroblasts, which are much easier to adapt to long-term tissue culture, have been targeted in some cases. For example, transfer of genes encoding the cytokines IL-2 and IL-4 into skin fibroblasts grown in culture provides the basis of some clinical trials for treatment of breast cancer, colorectal cancer, melanoma and renal cell carcinoma. The IL-2- and IL-4-secreting fibroblasts are then mixed with irradiated autologous tumor cells and injected subcutaneously. In such cases, the hope is that the local production and secretion of cytokines by the transferred fibroblasts will induce a vigorous immune response to the nearby irradiated tumor cells and thereby result in a systemic anticancer immune response.

Other immunological approaches

Two other ex vivo gene therapy strategies use immunological approaches to tumor destruction. One involves transferring an antisense insulin-like growth factor-1 (IGF1) gene into tumor cells in order to block production of IGF-1 (Anthony et al., 1998). Animal studies have shown that when tumor cells modified in this way are reimplanted in vivo, they provoke an immune response which can lead to destruction of nonmodified tumors, but the basis of immunological destruction is not known. A second approach involves the insertion of a co-stimulatory molecule such as B7-1 or B7-2, molecules which are normally present on lymphocytes, being required for full T-lymphocyte activation (see Putzer et al., 1997).

In vivo gene therapy approaches for cancer

In vivo gene therapy may be the only feasible approach for some cancers

Currently, a variety of different gene therapy approaches are being used involving genetic modification of tumor cells in vivo. In some cases, adoptive immunotherapy approaches are being employed, as in the case of increasing the immunogenicity of melanoma, colorectal tumors and a variety of solid tumors by the direct injection of liposomes containing a gene which encodes HLA-B7. The tumor cells take up the liposomes by phagocytosis and express the foreign HLA-B7 antigen transiently on their cells. More recent modifications include the additional insertion of a gene encoding the conserved light chain of HLA antigens, β2-microglobulin.

A second approach has been the use of retrovirus-mediated transfer of a gene encoding a prodrug, a reagent that confers sensitivity to cell killing following subsequent administration of a suitable drug. In one recent example, the target cells were brain tumor cells, notably recurrent glioblastoma multiforme, and the retroviruses were provided in the form of murine fibroblasts that are producing retroviral vectors (retroviral vector-producing cells or VPCs). The cells were directly implanted into multiple areas within growing tumors using stereotactic injections guided by magnetic resonance imaging. Once injected, the VPCs continuously produce retroviral particles within the tumor mass, transferring genes into surrounding tumor cells. Although retroviruses are not normally used for in vivo gene therapy because of their sensitivity to serum complement, they are comparatively stable in this special environment and have the advantage that, since they only infect actively dividing cells, the tumor cells are a target, but not nearby brain cells (which are usually terminally differentiated).

In vivo gene therapy for brain tumors

This example shows a strategy for treating glioblastoma multiforme in situ using a delivery method based on magnetic resonance imaging-guided stereotactic implantation of retrovirus vector-producing cells (VPCs). The retroviral vectors produced by the cells were used to transfer a gene encoding a prodrug, herpes simplex thymidine kinase (HSV-tk), into tumor cells. This reagent confers sensitivity to the drug gancyclovir: HSV-tk phosphorylates gancyclovir (gcv) to a monophosphorylated form gcv-P and, thereafter, cellular kinases convert this to gancyclovir triphosphate, gcv-PPP, a potent inhibitor of DNA polymerase which causes cell death. Because retroviruses infect only dividing cells, they infect the tumor cells, but not normal differentiated brain cells. The implanted VPCs transferred the HSV-tk gene to neighboring tumor cells, rendering them susceptible to killing following subsequent intravenous administration of gancyclovir. In addition, it was found that uninfected cells were also killed by a bystander effect: the gancyclovir triphosphate appeared to diffuse from infected cells to neighboring uninfected cells, possibly via gap junctions. Reproduced in part from Culver and Blaese (1994) with permission from Mary Ann Liebert Inc.

The prodrug gene that was transferred is a HSV gene which encodes thymidine kinase (HSV-tk). HSV-tk confers sensitivity to the drug gancyclovir by phosphorylating it within the cell to form gancyclovir monophosphate which is subsequently converted by cellular kinases to gancyclovir triphosphate. This compound inhibits DNA polymerase and causes cell death. Such therapy appears to benefit from a phenomenon known as the bystander effect: adjacent tumor cells that have not taken up the HSV-tk gene may still be destroyed. This is thought to be due to diffusion of the gancyclovir triphosphate from cells which have taken up the HSV-tk gene, perhaps via gap junctions.

Gene therapy for infectious disorders

Gene therapy for infectious disorders is often aimed at selectively interfering with the life-cycle of the infectious agen

Current gene therapy trials for infectious disorders are conspicuously targeted at treating AIDS patients. The infectious agent for this usually fatal disorder is a class of retrovirus known as HIV-1 which can infect helper T lymphocytes, a crucially important subset of immune system cells. Two features of HIV-1 make it especially deadly: it eventually kills the helper T cells (thereby rendering patients susceptible to other infections), and the provirus tends to persist in a latent state before being suddenly activated (the lack of virus production during the latent state complicates antiviral drug treatment). A major problem is that the HIV genome is mutating at a very high rate.

The HIV-1 virus life-cycle

The HIV-1 virus is a retrovirus which contains two identical single-stranded viral RNA molecules and various viral proteins within a viral protein core, which itself is contained within an outer envelope. The latter contains lipids derived from host cell plasma membrane during budding from the cell, plus viral coat proteins gp120 and gp41. Penetration of HIV-1 into a T lymphocyte is effected by specific binding of the gp120 envelope protein to the CD4 receptor molecules present in the plasma membrane. After entering the cell, the viral protein coat is shed, and the viral RNA genome is converted into cDNA by viral reverse transcriptase (RT). Thereafter a viral integrase ensures integration of the viral cDNA into a host chromosome. The resulting provirus (see top) contains two long terminal repeats (LTRs), with transcription being initiated from within the upstream LTR. For the sake of clarity, the figure only shows some of the proteins encoded by the HIV-1 genome. In common with other retroviruses, are the gag (core proteins), pol (enzymes) and env (envelope proteins) genes. Tat and rev are regulatory proteins which are encoded in each case by two exons, necessitating RNA splicing. The tat protein functions by binding to a short RNA sequence at the extreme 5′ end of the RNA transcript, known as TAR (trans-acting response element); the rev protein binds to an RNA sequence, RRE (rev response element), which is encoded by sequence transcribed from the env gene.

In principle, a variety of gene therapy strategies can be envisaged for treating AIDS. As in the case of cancer gene therapy, infected cells can be killed directly (by insertion of a gene encoding a toxin or a prodrug; see above) or indirectly, by enhancing an immune response against them. For example, this can involve transferring a gene that encodes an HIV-1 antigen, such as the envelope protein gp120, and expressing it in the patient in order to provoke an immune response against the HIV-1 virus, or the patient’s immune system can be boosted by transfer and expression of a gene encoding a cytokine, such as an interferon. Another general approach, which is applicable to all disorders caused by infectious agents, is to find a means of interfering with the life-cycle of the infectious agent.

Gene therapy strategies designed to interfere with the HIV-1 life-cycle

A wide variety of such strategies are available (Gilboa and Smith, 1994). Inhibition has been envisaged at three major levels:

  • Blocking HIV-1 infection. HIV-1 normally infects T lymphocytes by binding of the viral gp120 envelope protein to the CD4 receptor on the cell membrane. Transfer of a gene encoding a soluble form of the CD4 antigen (sCD4) into T lymphocytes or hemopoietic cells and subsequent expression will result in circulating sCD4. If the levels of circulating sCD4 are sufficiently high, binding of sCD4 to the gp120 protein of HIV-1 viruses could be imagined to inhibit infection of T-lymphocytes without compromising T lymphocyte function.
  • Inhibition at the RNA level. The production of HIV-1 RNA can be selectively inhibited by standard antisense/ribozyme approaches (see Section 22.3.2), and also by the use of RNA decoys. The latter strategy exploits unique regulatory circuits which operate during HIV replication. Two key HIV regulatory gene products are tat and rev which bind to specific regions of the nascent viral RNA, known as TAR and RRE respectively (Figure 22.14). Artificial expression of short RNA sequences corresponding to TAR or RRE will generate a source of decoy sequences which can compete for binding of tat and rev, and possibly thereby inhibit binding of these proteins to their physiological target sequences.
  • Inhibition at the protein level. There are numerous different strategies. One strategy involves designing intracellular antibodies (see Section 22.3.3), against HIV-1 proteins, such as the envelope proteins. Another involves introducing genes that encode dominant-negative mutant HIV proteins which can bind to and inactivate HIV proteins (transdominant proteins). For example, transdominant mutant forms of the gag proteins have been shown to be effective in limiting HIV-1 replication, possibly by interfering with multimerization and assembly of the viral core (Gilboa and Smith, 1994).