Gene Therapy & Neoplastic Disorders | Human Genetics

Let us make an in-depth study of the gene therapy principles for neoplastic disorders and infectious diseases.

Cancer Gene Therapies:

Many different approaches can be used for cancer gene therapy (see Culver and Blaese, 1994 and Table 23.6) and, in marked contrast to the few gene therapy trials for inherited dis­orders, numerous cancer gene therapy trials are currently being conducted (Table 23.7).

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—arti­ficially or by enhancing an immune response.

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.

Potential applications of gene therapy for the treatment of cancer

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 anti-tumor activity of immune system cells by, for example, inserting genes that encode cytokines

Induce normal tissues to produce anti-tumor 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

In principle, two types of cancer gene therapy strategy can be envisaged:

(i) Tumor Reduction Strategies:

Many current cancer gene therapy strategies are not expected to result in 100% success at targeting the tumor cells. For example, gene therapy based on simple gene trans­fer into tumor cells (to ensure direct killing of the cancerous cells) is, like any other current form of gene transfer, compara­tively inefficient and so some tumor cells may not be targeted. Accordingly, such types of treatment may be viewed as a refinement of conventional radiotherapy and surgical treatments.

(ii) Tumor Elimination Strategies:

Such approaches are intended to kill 100% of cancer cells. If, for example, immune sys­tem cells can be stimulated into a specific immune response against the tumor cells, complete remission may be possible. No matter which method is used, however, the aim of complete elimination of the cancer­ous cells may not be easy to attain because of the rapid evolution of cancer cells and strong selection for resistant cells.

Gene Therapy for Infectious Disorders:

The gene therapy approaches for treating infectious disorders are slightly different. In common with cancer gene therapy are the prin­ciples of provoking a specific immune response or specific killing of infected cells. In addition, and increasingly popular, are strategies that are intended to affect the life-cycle of the infectious agent, reducing its ability to undergo productive infection.

Some infectious agents are genetical­ly comparatively stable. Others, however, may be undergoing rapid evolution, and, much as in the case of cancer cells, present problems for any general therapy. The classic example is AIDS, where the infectious agent, HIV-1, appears to mutate rapidly.

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-a (TNF-α), was transferred into tumor-infiltrating lymphocytes (TILs) in an effort to increase their anti-tumor 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 (Fig. 23.13). 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.

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 colony- stimulating 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 re-implantation of non-modified 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 (see Table 23.7).

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 (see Table 23.7 for some examples). 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 lL-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 anti-cancer immune response.

Other Immunological Approaches:

Two other ex vivo gene therapy strategies are using immunological approaches to tumor destruction. One involves transferring an anti- sense insulin-like growth factor-1 (IGF1) gene into tumor cells in order to block production of lGF-1.

Animal studies have shown that when tumor cells modified in this way are re-implanted in vivo, they provoke an immune response which can lead to destruction of non-modified 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.

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 ‘pro drug’, 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 (Fig. 23.14). 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).

The pro drug 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 (see Fig. 23.14). Such ther­apy appears to benefit from a phenomenon known as the by-stander 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 is often aimed at selectively interfering with the life-cycle of the infectious agent:

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 (see Fig. 23.15).

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 anti-viral drug treatment). A major prob­lem is that the HIV genome is mutating at a very high rate.

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 pro-drug; see above) or indirectly, by enhancing an immune response against them.

For example, this can involve transferring a gene that encodes an HlV-1 antigen, such as the envelope protein gp 120, 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.